Universität zu Köln

Kolek, Susanne Dorothea ORCID: 0009-0004-0475-6048 (2026). Development of clippase-based tools for studying ubiquitin and ubiquitin-like modification systems. PhD thesis, Universität zu Köln.

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Abstract

The post-translational modification by the small modifier ubiquitin regulates virtually all cellular processes and is indispensable for protein homeostasis, cell proliferation and cell survival. In recent years, a plethora of mass spectrometry-based methodologies have been developed to elucidate ubiquitin targets, their individual modified sites and cellular ubiquitin chain architectures. Further, studies aimed to get a holistic insight into the modifications by the ubiquitin-like proteins Nedd8, ISG15 and SUMO. However, current studies employ either very complex strategies to discern between modifiers, which are often conflated due to their primary sequence similarities, or disregard their conflation altogether. The characterization of several members of a novel class of deubiquitinating enzymes of bacterial origin paved the way to utilize them in mass spectrometry-based studies of ubiquitin and ubiquitin-like modification systems. These enzymes ‘clip’ within the modifiers C-terminus, leaving a GlyGly-remnant at the modification sites. Due to their unusual mode of action, these enzymes were termed clippases. One of the characterized enzymes is BpJOS from Burkholderia pyrrocinia, which exhibits exceptionally high activity against ubiquitin while acting promiscuously on all ubiquitin chain types. On top, BpJOS is inert against the modifier ISG15. Based on the activity of BpJOS, two different mass spectrometry-based methodologies were developed to fill in the gaps left by the currently available, established protocols. First, the easy-to-apply method termed ‘Clippomics’ was conceptualized. This method allows for the distinguishment between ubiquitin and ISG15 sites in complex samples upon in-parallel treatment with the published ISG15-targeting clippase Lbpro. Moreover, BpJOS treatment alone enables specifically the identification of ubiquitin sites in presence of ISG15. By that, the shifts in the ubiquitome upon interferon stimulation, as occurring under infection conditions, were studied. Both applications serve as proof-of-concepts for their use in ensuing studies, as the ‘Clippomics’ method makes further genetic manipulation for the distinguishment of ubiquitin and ISG15 dispensable and can be used on a wide array of samples. Second, BpJOS was utilized for the investigation of ubiquitin chain linkages in in vitro studies. The ‘Ubi clipping with BpJOS’ methodology is inspired by the ‘Ub-clipping’ approach utilizing Lbpro. The clippase-generated GlyGly-remnants by treatment of ubiquitin chains allow for the in-depth characterization of present chain linkages and architectures. This process is optimized by the high activity of BpJOS against ubiquitin compared to the published Lbpro clippase. Taken together, BpJOS was identified as a powerful, multi-faceted tool for the elucidation of ubiquitin and ubiquitin-like modifications. Both developed methodologies harbor great potential to significantly contribute to the research field of modification systems.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Kolek, Susanne Dorothea UNSPECIFIED https://orcid.org/0009-0004-0475-6048 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-813119
Date:	2026
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Clippase; Ubiquitin; Deubiquitinase English
Date of oral exam:	29 May 2026
Referee:	Name Academic Title Hofmann, Kay Prof. Dr. Dohmen, Jürgen Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/81311