Monitoring protein synthesis and degradation by metabolic AHA labeling and click chemistry based detection

Abstract

The proteome determines the biochemical reactions that can be carried out by the cell, and is thus highly dynamic and tightly regulated by protein synthesis and degradation for adaption to environmental cues and cellular alterations. Cellular proteins are degraded by two main systems; the proteasomes and in the lysosomes. In this thesis a new approach for protein labeling has been established that allow quantification and imaging of protein synthesis and degradation. In this method, the amino acid analog L-Azidohomoalanine (AHA) replaces the amino acid methionine and is incorporated into proteins during translation. The labeled proteins containing AHA is further detected by a click chemistry based reaction. The established method has been employed to study the balance of protein synthesis and degradation (protein turnover), mainly focusing on autophagic degradation of labeled proteins. AHA labeled proteins have been detected by different approaches, including near infrared (NIR) detection, western blot analysis and immunofluorescent confocal imaging, in order to evaluate the possibilities and limitations for each detection method. A limitation of the approach was found for proteins that are difficult to extract from cells using conventional lysis buffers since such proteins are lost from lysates prior to click labeling. However, immunofluorescent confocal imaging of labeled proteins offered the possibility to visualize turnover of proteins and protein aggregates.

Stress and translational errors may produce misfolded, aggregate prone proteins that are toxic to cells. The initial phases of the neurodegenerative eye disease age-related macular degeneration (AMD) is associated with protein aggregation and impaired autophagy in retinal pigment epithelial (RPE) cells. To study protein turnover in RPE cells, the normal non-transformed RPE cell line ARPE-19 have been employed as a model system. Epidemiological studies have suggested a reduced risk for development of AMD by increased dietary intake of the omega-3 polyunsaturated (n-3 PUFA) docosahexaenoic acid (DHA). The cells were found to display high basal autophagy, which further increased after treatment with DHA. The protein synthesis inhibitor puromycin rapidly induced formation of cytoplasmic protein aggregates highly co-localized with p62 and conjugated ubiquitin, which were rapidly removed upon a chase. These observations suggest that the cells have an efficient system for formation and removal of protein aggregates. The turnover of AHA labeled proteins was studied to determine cellular responses towards DHA, in which DHA was found to increase the aggregation of AHA labeled puromycin induced protein aggregates. This observation suggests that increased aggregation of potentially harmful misfolded proteins followed by autophagic degradation may be a disease preventive mechanism initiated by DHA in early stages of the development of AMD. This new click chemistry based labeling technology serves as a powerful and versatile new tool for biomedical research.