New reactivities and functionalities introduced by mimics of protein post-translational modifications

Chen, Haoyu (2024): New reactivities and functionalities introduced by mimics of protein post-translational modifications. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Protein post-translational modifications (PTMs) play crucial roles in regulating protein function, stability, localization, and interactions. These modifications, including phosphorylation, glycosylation, ubiquitinoylation, acetylation, and methylation, among others, are able to activate or deactivate enzymatic activities, dictate subcellular localization, mediate protein-protein interactions, and label proteins for degradation. By dynamically altering protein properties, PTMs enable cells to respond rapidly to environmental changes and maintain homeostasis, thereby contributing to processes such as signal transduction, immune response, cell cycle control, and apoptosis. To study protein PTMs, scientists employ a variety of methods such as high-resolution tandem mass spectrometry that allows for the precise identification and quantification of PTMs by analyzing peptide fragments. Western blotting, using specific antibodies recognizing modified residues, is another common method to detect and analyze PTMs. These methods, combined with bioinformatics tools, offer comprehensive approaches to understanding the complex roles of PTMs in protein regulation. We started to investigate a newly discovered PTM called adenylylation (AMPylation) by developing halogen-modified AMPylation mimics for subsequent cross-coupling via Suzuki-Miyaura reactions in the living cells. However, the downstream chemical biology analysis using functionalized aryl pinacol boronates revealed an unknown reactivity. For example, incubation between cell lysates and a fluorescent aryl pinacol boronate has brought significant labelling regardless of the addition of artificial AMPylation probes or palladium catalysts. To our delight, we found that the unknown reactivity was due to selective protein labelling (S-arylation on disulfides) triggered by ammounium persulfate (APS). Then oxidized glutathione and two more short peptides were applied to verify the S-arylation product formation, which was further fragmented in high-resolution MS2 to identify diagnostic ions. The radical-based mechanistic pathway of this reaction was confirmed by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap experiments, and the reaction was found to proceed only when aryl moieties were substituted with electron-donating groups, which was consistent with competition studies in cell lysates. This method was the first study to perform selective disulfide S-arylation with aryl radicals in mild aqueous conditions, and there was no need to use metal catalyst or photocatalysis. On the other hand, we took advantage of protein glycosylation and ubiquitinoylation to develop a two-component proteolysis targeting chimeras (PROTACs) strategy, which was proved to be able to selectively target O-GalNAcylated and O-GlcNAcylated proteins for proteasomal degradation. As a result, the critical metabolic and signaling pathways governed by glycoproteins were heavily perturbated, triggering severe cytotoxicity in human cancer cell lines. The approach termed GlyTACs leveraged from metabolic incorporation of easily accessible and cell-permeable peracetylated N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) mimics bearing an azide group into glycoproteins. In the living cells, the azido-modified glycoproteins served as covalent anchors for the introduction of thalidomide moiety by strain-promoted azide-alkyne cycloaddition (SPAAC) to recruit E3 ligase cereblon, resulting in stepwise ubiquitinoylation of ‘sensitized’ proteins and their degradation by proteasome. The efficiency of the GlyTAC system was shown in a series of human cancer cell lines and the mechanistic pathway was verified by performing control experiments at each stage of the process. Given the characteristic features of cancer cells including fast nutrient turnover, and overall increase of protein glycosylation, as well as the low cytotoxicity of the individual components, this GlyTAC approach may open a feasible strategy in cancer therapy. In summary, these studies have discovered unexplored reactivities and functionalities of protein PTMs, helping chemical biologists utilize bioorthogonal reactions by avoiding unwanted side reactions and introducing therapeutic potentials.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. Oktober 2024
• 1. Bericht­erstatter:in: Kielkowski, Pavel