Project No. 2128

Primary Supervisor

Dr. Jerome Korzelius – University of Kent

Co-Supervisor(s)

Dr. Paul Skipp – University of Southampton

Dr. Alessandro Ori – Research group leader, FLI/Leibniz Institute on Ageing, Jena, Germany

Summary

Ageing involves the functional decline of cells, tissues and organs over time, leading to disease and death.

Loss of proteostasis, the balance between the synthesis and degradation of proteins, is a hallmark of ageing and plays a causal role in many age-associated diseases, such as Alzheimer’s and Parkinson’s disease.

The protein complex known as the proteasome plays a key role in maintaining proteostasis via selective degradation of damaged and unwanted proteins. Our recent work highlighted a crucial role of the proteasome in brain ageing. However, how proteasome composition and its interaction with other proteins change with aging remains largely unknown. Another open question is whether the proteasome functions differently between organs during ageing, especially organs that can regenerate during life, e.g. the gut, and those that have limited regenerative capacity such as the brain.

In this project, we will monitor proteasome composition and interactions in vivo in the fruit fly (Drosophila melanogaster). We will implement an approach based on proximity labelling coupled to quantitative mass spectrometry. This technique uses an engineered biotin-ligase (e.g., BirA*) fused to a core proteasome component to label, in living cells, all proteasome-interacting proteins with biotin. Subsequent pulldown of biotinylated proteins and identification by mass-spectrometry enables the quantitative monitoring of the proteasome interactome.

We will express a proteasome-BirA* fusion protein under control of the Gal4/UAS inducible gene expression system to target it to both brain and intestine. From these tissues, we will quantify proteasome-interacting proteins from young, middle and old-aged flies. This will tell us how the stoichiometry of the proteasome and its interactions change over time and between tissues. Finally, we will take advantage of the excellent genetic tools and short lifespan of Drosophila to address the role of age-affected proteasome-interacting proteins on lifespan by using brain- and intestine-targeted knockdown by RNAi and lifespan assays.