Research focus of the Groettrup group
1) The biochemistry and function of FAT10 - an MHC encoded and cytokine inducible ubiquitin-like protein
FAT10 (HLA-F-adjacent transcript 10) was identified by sequencing of the human MHC class I locus and belongs to the family of the ubiquitin-like modifiers. FAT10 is a protein of 18 kDa consisting of two ubiquitin-like domains in a head to tail arrangement, separated by a short linker (Fig. 1).
Although it was initially reported to be expressed in mature B cells and dendritic cells only, FAT10 can be synergistically induced in cell lines from virtually all tissues with the proinflammatory cytokines IFNγ and TNFα. Interestingly, induction of FAT10 expression leads to cell death by apoptosis within 2 days. Furthermore FAT10 is up-regulated in cancers of liver and colon probably due to the IFNγ and TNFα production of the tumor environment and it plays a role in the regulation of chromosomal stability. FAT10 can be covalently conjugated to so far unidentified target proteins via its C-terminal diglycine motif and FAT10ylated proteins become degraded by the proteasome or are transported to aggresomes. Currently we are characterizing the enzymes involved in the FAT10 conjugation pathway and characterize previously identified substrates of FAT10ylation. Moreover, we study the function of FAT10 in thymic T cell selection, in immune regulation, and defence against pathogens using FAT10 deficient mice.
Fig. 1: Model of the structure of FAT10; the two ubiquitin-like domains of FAT10 are connected by a short linker [modified from Groettrup et al. (2008) Trends Biochem Sci 33:230-237].
Selected publications from this project:
Buerger, S., Herrmann, V.L., Mundt, S., Trautwein, N., Groettrup, M., and Basler, M. (2015) The ubiquitin-like modifier FAT10 is selectively expressed in medullary thymic epithelial cells and modifies T cell selection. J. Immunol. 195: 4106-4116.
Spinnenhirn, V., Farhan, H., Basler, M., Aichem, A., Canaan, A., Groettrup, M. (2014) - The ubiquitin-like modifier FAT10 decorates autophagy targeted Salmonella and contributes to resistance of mice. J Cell Sci. Nov 15;127(22):4883-93
Aichem A, Kalveram B, Spinnenhirn V, Kluge K, Catone N, Johansen T, Groettrup M. (2012) - The proteomic analysis of endogenous FAT10 substrates identifies p62/SQSTM1 as a substrate of FAT10ylation. J Cell Sci. 2012 Oct 1;125(Pt 19):4576-85. doi: 10.1242/jcs.107789. Epub 2012 Jul 13.
Aichem A, Pelzer C, Lukasiak S, Kalveram B, Sheppard PW, Rani N, Schmidtke G, Groettrup M. (2010) - USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis. Nat Commun. 2010 May 4;1:13. doi: 10.1038/ncomms1012.
Rani N, Aichem A, Schmidtke G, Kreft SG, Groettrup M. (2012) - FAT10 and NUB1L bind to the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis. Nat Commun. 2012 Mar 20;3:749. doi: 10.1038/ncomms1752.
Pelzer C, Groettrup M. (2010) - FAT10 : Activated by UBA6 and Functioning in Protein Degradation. Subcell Biochem. 2010;54:238-46. doi: 10.1007/978-1-4419-6676-6_19.
Schmidtke G, Kalveram B, Groettrup M. (2009) - Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L. FEBS Lett. 2009 Feb 4;583(3):591-4. doi: 10.1016/j.febslet.2009.01.006. Epub 2009 Jan 21.
Kalveram B, Schmidtke G, Groettrup M. (2008) - The ubiquitin-like modifier FAT10 interacts with HDAC6 and localizes to aggresomes under proteasome inhibition. J Cell Sci. 2008 Dec 15;121(Pt 24):4079-88. doi: 10.1242/jcs.035006. Epub 2008 Nov 25
Hipp MS, Raasi S, Groettrup M, Schmidtke G. (2004) - NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation. J Biol Chem. 2004 Apr 16;279(16):16503-10. Epub 2004 Feb 2
Hipp MS, Kalveram B, Raasi S, Groettrup M, Schmidtke G. (2005) - FAT10, a ubiquitin-independent signal for proteasomal degradation. Mol Cell Biol. 2005 May;25(9):3483-91
Raasi S, Schmidtke G, Groettrup M. (2001) - The ubiquitin-like protein FAT10 forms covalent conjugates and induces apoptosis. J Biol Chem. 2001 Sep 21;276(38):35334-43. Epub 2001 Jul 9
2) Development of an immune therapy for prostate cancer
Prostate carcinoma (CaP) is becoming an increasingly severe health problem for men in industrialized countries. Due to an enhanced life expectancy and a more intensified clinical scrutiny, CaP has become the most frequently diagnosed cancer in men. The probability of disease is 10% and the risk of dying from CaP is 3%, i.e. about 40'000 men die from CaP every year.
After the formation of metastases, carcinoma growth can be suppressed during several months by hormone ablation therapy in most cases. Unfortunately, male sex hormone-independent tumor growth develops in most cases after 1-2 years. There is no effective therapy for these hormone refractory CaP tumors till now, and even the newest achievements in chemotherapy reveal modest life-prolonging effects only. For that reason, we have been developing a prostate cancer immune therapy for many years now. The aim of this immune therapy is a systematic fortification of the patient's own immune defence against the carcinoma in a way that helps the recognition and killing of tumor cells by so-called T killer cells. A big advantage of immune therapy is the high specificity for tumor cells, whereas only minor side effects are expected to occur. Another advantage is that immune cells patrol the body and are thus able to find and eliminate distant metastases. At present immune therapies are in development for clinical trials but the techniques still have to be improved in order to evoke strong enough immune responses in patients. Up to now success in immune therapy is still restricted to a minority of patients. We conducted a phase I clinical trial in collaboration with the Departments for Oncology and Urology at the Cantonal Hospital of St. Gallen using dendritic cells (DC) grown from patients' blood that were loaded with tumor antigens and re-injected into the patients. Using this technique, immune responses against tumor antigens were induced in some patients resulting in attenuation of disease progression. However, DC generation is laborious and expensive, rendering a clinical application on a large scale rather improbable.
At the BITg, we are looking for new strategies to deliver tumor antigens to patients' DC by subcutaneous injection in a way that they can be readily taken up by the DC. A laborious cultivation of DC could thus be avoided. In collaboration with the Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich (Prof. Dr. Bruno Gander), we are developing techniques to improve the encapsulation of tumor antigens together with DC maturation stimuli into biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (Fig. 2). The clinically approved material for the production of these microspheres is also used in the production of self dissolving surgical suture.
Fig. 2: Electron microscopic image of biodegradable poly(D,L-lactide-co-glycolide) microspheres.
After microsphere uptake (Fig. 3) ), DC migrate into lymph nodes (please refer to the projects of Daniel Legler's group), where they stimulate tumor combating T lymphocytes. We could already demonstrate that microsphere uptake by DC does not alter their biological properties and that incorporated antigens can be presented on MHC class I as well as MHC class II molecules over a prolonged period of several days.
Fig. 3: Immature human dendritic cells loaded with PLGA microspheres (image: Groettrup lab). The cells were incubated with PLGA microspheres for four hours and subsequently monitored by phase contrast microscopy.
Selected publications from this project:
Herrmann, V.L., Wieland, D.E., Legler, D.F., Wittmann, V., Groettrup, M. (2016)The STEAP1262-270 Peptide Encapsulated into PLGA Microspheres Elicits Strong Cytotoxic T Cell Immunity in HLA-A*0201 Transgenic Mice – a New Approach to Immunotherapy against Prostate Carcinoma. Prostate. 76(5):456-68.
Herrmann VL, Hartmayer C, Planz O , Groettrup M. (2015) Cytotoxic T cell vaccination with PLGA microspheres interferes with influenza A virus replication in the lung and suppresses the infectious disease. J Control Release Aug 12;216:121-131. doi: 10.1016/j.jconrel.2015.08.019
Mueller M, Reichardt W, Koerner J and Groettrup M. (2012). Coencapsulation of tumor lysate and CpG-ODN in PLGA-microspheres enables successful immunotherapy of prostate carcinoma in TRAMP mice. J Control Release, 162(1): 159-66.
Mueller M, Schlosser E, Gander B , Groettrup M. - (2010) 'Tumor eradication by immunotherapy with biodegradable PLGA microspheres - an alternative to incomplete Freund's adjuvant' Int. J. Cancer 129(2): 407-16.
Fischer S, Schlosser E, Mueller M, Csaba N, Merkle HP, Groettrup M, Gander B. (2009). - Concomitant delivery of a CTL-restricted peptide antigen and CpG ODN by PLGA microparticles to induce cellular response. - J Drug Target, 17(8): 652-61.
Schlosser E, Mueller M, Fischer S, Basta S, Busch DH, Gander B, Groettrup M. – 2008. – TLR ligands and antigen need to be coencapsulated into the same biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses. – Vaccine 26(13):1626-1637.
Fischer, S., Uetz-von Allmen, E., Waeckerle-Men, Y., Groettrup, M., Merkle, H. P., Gander, B. (2007). Dendritic cells preserve their phenotype and functionality upon phagocytosis of polyelectroclyte-coated PLGA microparticles. Biomaterials 28: 994-1004.
Waeckerle-Men, Y., Uetz-von Allmen, E., Gander, B., Scandella, E., Schlosser, E., Schmidtke, G., Merkle, H. P., and Groettrup, M. (2006). Encapsulation of proteins and peptides into biodegradable poly(D,L-lactide-co-glycolide) microspheres prolongs and enhances antigen presentation by human dendritic cells. Vaccine: 24, 1847-1857.
Waeckerle-Men, Y., Scandella, E., Uetz-von Allmen, E., Ludewig, B., Gillessen, S., Merkle, H. P., Gander, B., and Groettrup, M. (2004). Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable PLGA microspheres for DC-vaccination. J. Immunol. Methods: 287, 109-124
3) The inhibition of the immunoproteasome as therapeutic approach against autoimmunity and cancer
The immunoproteasome is a large cylindrical complex in immune cells in which porteins are degraded to peptides (Fig. 4). So far the immunoproteasome had been shown to be involved in the fragmentation of antigens for the stimulation of cytotoxic T lymphocytes.
Fig. 4: Crystal structure of the mouse immunoproteasome [from Huber et al. (2012) Cell 148:727-738].
In 2009 we have discovered a new function of the immunoproteasome in the development of pro-inflammatory T helper lineages (Th1, Th17) and cytokines (IL-6, IFN-gamma, IL-23, TNF, see Muchamuel et al. (2009) Nature Medicine 15:781-787). These T helper cells and cytokines are critically involved in the pathogenesis of autoimmune diseases. We could show that an inhibitor of the immunoproteasome subunit LMP7 protected from the development and exacerbation of several autoimmune diseases (multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis) in preclinical models. Moreover, the development and progression of colon carcinoma could be blocked with immunoproteasome inhibition. Based on our research, several pharmaceutical companies have developed novel immunoproteasome inhibitors that are currently tested in clinical trials as therapeutics against autoimmune diseases.
Koerner, J., Brunner, T., and Groettrup, M. (2017) Inhibition and deficiency of the immunoproteasome subunit LMP7 suppress the development and progression of colorectal carcinoma in mice. Oncotarget. doi: 10.18632/oncotarget.15141.
Basler M*, Mundt S*, Muchamuel T*, Moll C, Jiang J, Groettrup M, Kirk CJ. (2014) Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis. EMBO Mol Med 6(2):226-238.
Basler M, Kirk CJ, Groettrup M. (2013) The immunoproteasome in antigen processing and other immunological functions. Curr Opin Immunol, 2013;25(1):74-80.
Huber EM*, Basler M*, Schwab R, Kirk CJ, Heinemeyer W, Groettrup M, and Groll M. (2012). Immuno- and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity. Cell: 148(4), 727-738.
Kalim KW, Basler M, Kirk CJ, Groettrup M. (2012). - Immunoproteasome subunit LMP7 deficiency and inhibition suppresses Th1 and Th17 but enhances regulatory T cell differentiation J Immunol 189(8):4182-4193.
Muchamuel T*, Basler M*, Aujay MA, Suzuki E, Kalim KW, Lauer C, Sylvain C, Ring ER, Shields J, Jiang J, Shwonek P, Parlati F, Demo SD, Bennett MK, Kirk CJ, and Groettrup M. (2009). - A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. - Nat. Med.: 15(7), 781-7.
Basler M, Moebius J, Elenich L, Groettrup M, and Monaco JJ. (2006). An altered T cell repertoire in MECL-1-deficient mice. J Immunol.: 176(11), 6665-6672.
Groettrup M, Kirk CJ, Basler M. (2010). - Proteasomes in immune cells: more than peptide producers? - Nature Rev. Immunol. 10(1):73-8.
Selected publications from this project: