Chemical Biology of Protein Post-translational Modifications

Neurological disorders and in particular neurodegeneration is becoming ubiquitous in ageing populations. Regardless of many efforts, the fundamental principles underlying the mechanisms of failing neurodevelopment and onset of the neurodegeneration are still unknown. The protein post-translational modifications (PTMs) are closely connected to multiple metabolic and signalling pathways involved in neurodegeneration.

Our research focuses on development of chemical biology tools to analyse protein post-translational modifications. We combine induced pluripotent stem cells-based cellular models, quatitative mass spectrometry techniques and organic synthesis to track and visualize PTMs in living cells.

Protein AMPylation

Recently, our group has identified the Alzheimer's disease associated protein PLD3 to be specifically AMPylated in lysosomal lumen of young and mature neurons. We have established a novel gel-based methodology enabling the separation of the modified and unmodified proteins complementing the mass spectrometry data. Combination of both has revealed fascinating processing pathway of the PLD3 during neuronal differentiation and maturation.

Becker, T.; Wiest, A.; Telek, A.; Bejko, D.; Hoffman-Röder, A.; Kielkowski, P.: "Transforming chemical proteomics enrichment into high throughput method using SP2E workflow." JACS Au, 2022 DOI: 10.1021/jacsau.2c00284.

Becker, T.; Cappel, C.; Di Matteo, F.; Sonsalla, G.; Kaminska, E.; Spada, F.; Cappello, S.; Damme, M.; Kielkowski, P.: "AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins." iScience, 2021, 24, 103521. DOI: 10.1016/j.isci.2021.103521.

Sieber, S. A.; Cappello, S.; Kielkowski, P.: "From young to old: AMPylation hits the brain." Cell Chem. Biol., 2020, 27, 773-779.

Protein Tyrosination

Tubulin detyrosination-tyrosination cycle regulates the stability of microtubules. Thus far described on α-tubulins, the tyrosination level is maintained by a single tubulin-tyrosine ligase (TTL). However, the precise dynamics and tubulin isoforms which undergo (de)tyrosination in neurons are unknown. Here, we exploit the substrate promiscuity of the TTL to introduce an O-propargyl-L-tyrosine in neuroblastoma cells and neurons. Mass spectrometry-based chemical proteomics in neuroblastoma cells using the O-propargyl-L-tyrosine probe revealed previously discussed tyrosination of TUBA4A, MAPRE1, and other non-tubulin proteins. This finding was further corroborated in differentiating neurons. Together we present the method for tubulin tyrosination profiling in living cells. Our results show that detyrosination-tyrosination is not restricted to α-tubulins with coded C-terminal tyrosine and is thus involved in fine-tuning of the tubulin and non-tubulin proteins during neuronal differentiation.

Makarov, D.; Kielkowski, P.: "Chemical proteomics reveals protein tyrosination beyond the alpha-tubulins in human cells." ChemBioChem, 2022, DOI: 10.1002/cbic.202200414.

Chemical Proteomics

To analyse protein PTMs we utilize mass spectrometry-based chemical proteomic approach. With the increasing evidence of protein PTMs crucial role in neurological disorders, we are interested in developing innovative chemical proteomic strategies to facilitate unambiguous analysis of protein PTMs in different cell types of central nervous system.

Makarov, D.; Telek, A.; Becker, T.; von Wrisberg, M.; Schneider, S.; Kielkowski, P.: "Clickable report tags for identification of modified peptides by mass spectrometry." J. Mass Spectrom., 2022, 13, e4812. DOI: 10.1002/jms.4812.

Kielkowski, P.; Buchsbaum, I.; Kirsch, V.; Bach, N.; Drukker, M.; Cappello, S.; Sieber, S.: "FICD activity and AMPylation remodelling modulate human neurogenesis." Nat. Commun., 2020, 11, 517.

Induced Pluripotent Stem Cells

We are convinced that human induced pluripotent stem cells possess a new hope for deciphering of molecular mechanisms behind neurological disorders.