NeuroProClick · Protein-engineering based approach to detect oxidative stress-induced changes in the neuronal proteome

Protein-engineering based approach to detect oxidative stress-induced changes in the neuronal proteome

Neurodegenerative disorders are characterised by the disruption of key cellular processes and neuronal loss. Injury via oxidative stress is also associated with the progression of these diseases. Identifying changes within neuronal cells at specific time points will help us better understand the role of oxidative stress in these diseases. These cellular changes are mediated by biomolecules known as proteins, which serve a variety of roles within cells, including structural support, catalysis, transport, and signalling. Quantitative proteomics refers to techniques used to determine protein amounts in biological samples. Such methods have been used to detect proteome changes in immortalised neuronal cells, primary cultured neurons and brain tissue. However, existing quantitative proteomic methods face limitations when examining proteome changes during specific time windows and in specific cells. One method that can address these limitations is genetic code expansion (GCE). Normally, a cell’s genetic code is restricted to 20 standard and two rare amino acids (AAs). An expanded genetic code uses non-canonical amino acids (ncAAs) to give proteins novel properties. Like canonical AAs, these ncAAs need translational machinery that adds them into proteins in response to codons defined by the genetic code. Therefore, cells need orthogonal translational machinery specific for the ncAA and a certain codon. Usually, the amber stop codon is reprogrammed to introduce ncAAs into individual proteins. Alternatively, sense codons can be targeted, so the ncAA can be added across the whole proteome. This strategy has been exploited for proteomic studies. This proteome-wide approach was first used in combination with bioorthogonal click chemistry for quantitative studies of newly synthesised proteins in fruit fly models. In this approach, the ncAAs were incorporated across the proteome, and bioorthogonal click chemistry, which refers to selective chemical reactions that occur under physiological conditions, was used to add a chemical handle for downstream enrichment and analysis. Incorporating these ncAAs can be timed to capture proteins synthesised after external factors and specific cell types or signalling pathways can be targeted. So far, this technology has mainly been used at the proof-of-concept level and the number of studies in neurons is limited. The main aim of this project was to develop a methodology using this GCE-based approach for precise investigations of neuronal proteome changes. We aimed to use GCE to incorporate clickable ncAAs proteome-wide at defined time points relative to an oxidative injury to examine proteome changes in immortalised cell lines and human-derived neurons.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз