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Projekt Druckansicht

Lysinacylierung in zellulärer Regulation, Alterung und Krankheiten.

Fachliche Zuordnung Biochemie
Biophysik
Strukturbiologie
Förderung Förderung von 2017 bis 2021
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 389564084
 
Erstellungsjahr 2022

Zusammenfassung der Projektergebnisse

Lysine acetylation is a post-translational modification (PTM) that was shown to be distributed in all domains of life in proteins covering all essential cellular functions. It is only incompletely understood how lysine acetylation regulates protein function and how its dysfunction contributes to the ageing process and how it supports the development of diseases. This research proposal applies a dual systemic and a targeted approach. In the systemic approach we aimed at determination of lysine acetylation stoichiometry on a systemic scale by quantitative mass-spectrometry. This is important to judge the physiological significance of individual lysine acetylation events. We initiated studies to assess the acetylation stoichimetry of liver tissues of mice at different age (young, middle-aged and old) by quantitative mass-spectrometry. We established a MS workflow including a chemical labelling step with acetoxy-d3-succinimide that allows to systemically determine the lysine acetylation stoichiometry. We faced problems with the labelling as we only obtained a labelling efficiency of app. 72%. As we could not solve the underlying problem, we switched to a cellular system. We analysed the acetylome of macrophages infected with S. typhimurium as infections were shown to result in downregulation of SRIT1 expression (with Dr. N. Robinson/University of Cologne). The labelling efficiency was almost 100%. The data is measured and evaluation of the data is ongoing. We expect to identify new interesting candidate proteins that are regulated by lysine acetylation, which will be analyzed further (will be published soon). We contributed to a study led by Dr. Brian Weinert/University of Copenhagen in which we analyzed the systemic acetylation stoichiometry of cervical cancer cells (HeLa cells). This study revealed that most high-stoichiometry sites are in nuclear proteins and sites that are enzymatically catalyzed (published in Nat. Commun.)8. In a targeted approach we aimed at development of a M. barkeri PylRS/tRNACUA-based system that allows to genetically encode succinyl-lysine or malonyl-lysine into proteins. These lysine modifications convert the positive charge of the lysine side chain into a negative suggesting functional differences compared to lysine acetylation. We established a selection strategy for a PylRS variant from a library (MEG/Eurofins). However, we were not able to obtain variants with the desired activities. Conducting lipidomics experiments (with Dr. S. Brodesser/University of Cologne) we could not confirm cellular uptake of the negatively charged succinyl-lysine. To this end, we synthesized succinyl-/malonyl-lysine ethyl-esters to neutralize the negative charges (with Prof. H.-G. Schmalz/University of Cologne) and performed screening experiments. However, also this approach was not successful. Future screening experiments might apply cell-free translation systems to circumvent the necessity of cellular uptake of the amino acids. Another objective was to structurally characterize sirtuin-substrate complexes. So far, no structure of a sirtuin-substrate complex is known. Our laboratory showed that some sirtuins can have a remarkable substrate specificity using site-specifically and natively-folded lysine-acetylated (AcK) proteins. As a strategy to stabilize transient enzyme-substrate complexes, we wanted to use proteins that are lysine trifluoro (TFAcK)- or thioacetylated (ThioAcK) as these AcK analogues are poor sirtuin substrates. We were able to identify a combination of a PylRS-variant and tRNACUA that most efficiently allows the incorporation of AcK, TFAcK and ThioAcK. We are in the process to produce proteins in yields and purity sufficient to perform biochemical and biophysical studies (manuscript will be published in the near future). We want to develop mechanism-based substrate derived peptides/proteins as potent and selective sirtuin inhibitors for therapeutic applications. Sirtuins were shown to contribute to disease development. Different sirtuin isoforms play context dependent different roles during disease development. To this end, the development of selective and potent sirtuin inhibitors is of great interest. We reported that RhoGDIa is specifically deacetylated at AcK52 by SIRT2. A modified trifluoroacetylated 13-mer peptide of RhoGDIa was capable to impair cell proliferation of cervical cancer (HeLa)-cells (published in ChemMedChem)16. Future studies will also include the structural component as we showed that this is important to determine sirtuin substrate specificity. Although several aspects of this proposal are not published yet, we made progress in all of the objectives of the research grant proposal. Several publications will follow in the next months.

Projektbezogene Publikationen (Auswahl)

 
 

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