Biochemical and functional characterization of ubiquitin-like proteines ISG15 and FAT10 in human dendritic cells.
Final Report Abstract
Post-translational modifications of proteins by either ubiquitin and or ubiquitin-like proteins (Ubls) are important regulatory principles in the adaptation of cells to changing physiological requirements. Conjugation of ubiquitin-like proteins often influences processes connected with the adaptation of cells to changing physiological requirements. Two cytokine induced ubiquitin-like proteins whose functions seem to be closely related to the immune response are ISG15 and FAT10. In the funded project we set out to analyze their regulation and/or function upon cytokine induction, their role in MHC class I antigen presentation as well in cross presentation by human dendritic cells (DC). We established that FAT-10–conjugation of substrates represents an alternative mode of post-translational protein modification that targets protein to proteasome dependent processing thereby contributing to MHC class I antigen presentation. FAT-10 distinguished itself from ubiquitin in that it promoted a HCMV-pp65(495-503) epitope triggered immune response that was dependent on proteasome subunit RPN10 and NUB1/NUB1L but was not influenced by immunoproteasomes and PA28. In addition, FAT10-conjugation to pp65 was also demonstrated to improve crosspresentation by human dendritic cells. Collectively, our experiments discovered the FAT10- NUB1/ NUB1L-Rpn10 axis as a novel route for MHC class I antigen direct presentation and also DC-based cross-presentation. Analysis of ISG15 conjugate formation revealed that the ISG15 E2 enzyme UBE2L6 was essentially responsible for the formation of ubiquitin– conjugates at early time points after type 1 IFN induction in the absence of ISG15. This changed at later time points with increasing amounts ISG15. Interestingly, IFN-γ was also found to induce ISGylation of protein-substrates. However, this conjugation did not involve the E3 ligase Herc 5. In siRNA screens and siRNA experiments we identified the E3 ligases UBE3A and ARIH1 as potential new ISG15 ligases. Our experiments also provided first evidence that the receptor CXCR4 facilitated the uptake of both FAT-10 and di-ubiquitin into dendritic cells thereby improving proteasome dependent cross presentation. Whether this is due to a direct or indirect involvement of CXCR4 in substrate uptake and processing remains to be shown.
Publications
- Maturation of human dendritic cells is accompanied by functional remodelling of the ubiquitin-proteasome system. Int J Biochem Cell Biol. 41,1205-15 (2009)
Ebstein, F., Lange, N., Urban, S., Seifert, U., Krüger, E., Kloetzel, P.M.
- Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell 142, 613-622 (2010)
Seifert, U., Bialy, L.P., Ebstein, F., Bech-Otschir, D., Voigt, A., Schröter, F., Prozorovski, T., Lange, N., Steffen, J., Rieger, M., Kuckelkorn., U, Aktas, O., Kloetzel, P.M., Krüger, E.
- Emerging roles of immunoproteasomes beyond MHC class I antigen processing. Cell.Mol.Life.Sci. 69, 2543-2558 (2012)
Ebstein, F., Kloetzel P.M., Krüger, E., Seifert, U.
(See online at https://doi.org/10.1007/s00018-012-0938-0) - The FAT10- and ubiquitin-dependent degradation machineries exhibit common and distinct requirements for MHC class I antigen presentation. Cell Mol Life Sci. 69, 2443-54 (2012)
Ebstein F, Lehmann A, Kloetzel PM
(See online at https://doi.org/10.1007/s00018-012-0933-5) - Immunoproteasomes are important for proteostasis in immune responses. Cell 152, 935-7 (2013)
Ebstein, F., Voigt, A., Lange, N., Warnatsch, A., Schröter, F., Prozorovski, T., Kuckelkorn, U., Aktas, O., Seifert, U., Kloetzel, P.M., Krüger, E.
