Dissecting the sugar-specificity of nucleocytoplasmic mono-glycosylation with AI designed protein tools

• Applicant: Dr. Martin Bonin, Ph.D.
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 569146282

In animals, thousands of intracellular proteins are modified by the addition of a single sugar called N-acetylglucosamine (GlcNAc). This modification, known as O-GlcNAcylation, is dynamically regulated by the enzymes O-GlcNAc transferase (OGT), which attaches GlcNAc, and O-GlcNAcase (OGA), which removes it. These modifications play crucial roles in various cellular processes, including transcription, translation, signal transduction, and metabolism. Despite their importance, the specific functions of these modifications remain poorly understood. Interestingly, similar systems exist in plants and yeasts, where different sugars are used for analogous modifications. In plants, proteins are modified with O-GlcNAc or O-fucose, while yeasts utilize O-mannose. These modifications are essential for development, growth and viability across different organisms. A key question is whether these modifications act as sugar-specific regulators or are functionally interchangeable. To address this question, I will create an OGT variant capable of transferring glucose instead of GlcNAc and introduce this variant into the fruit fly Drosophila melanogaster, replacing all O-GlcNAc with O-glucose. By analyzing the phenotypes of these genetically modified flies, I aim to determine if and which cellular pathways are affected by this sugar exchange. This approach will provide insights into the functional specificity of these modifications. In addition, I will use AI-based protein design to develop novel tools for detecting O-glucose modifications. These tools will be crucial for studying the modified proteins and understanding the broader implications of this sugar substitution. By determining whether downstream regulatory mechanisms are sugar-specific, this project challenges existing hypotheses, such as proposed sugar-specific O-GlcNAc readers. The results of this project will change our thinking on this important regulatory protein modification and have implications for the many diseases that are associated with O-GlcNAc dysregulation like Alzheimer’s and Parkinson’s disease. Ultimately, this project aims to reshape our understanding of this protein glycosylation and its role in cellular regulation.

• DFG Programme: WBP Fellowship
• International Connection: Denmark
• Host: Professor Dr. Daan van Aalten