Azidohomoalanine (AHA) Metabolic Labeling Reveals Unique Proteomic Insights into Protein Synthesis and Degradation in Response to Bortezomib Treatment

Background: Multiple myeloma (MM) is essentially an incurable Cancer, but treatments with Proteasome inhibitors are widely used clinically to extend patient survival. While the mechanisms of Proteasome inhibition by Bortezomib are well known, the cellular responses to this proteotoxic stress that leads to sensitivity by MM are not fully elucidated. This study reports on the application of an emerging method to investigate proteostasis by proteomics.

Methods: We utilized metabolic labeling with azidohomoalanine (AHA) in a MM cell line in combination with Bortezomib treatment. AHA labeling facilitates the selective isolation and identification of proteins for investigations of protein synthesis or protein degradation.

Results: The data collected reveals significant changes in gene protein synthesis upon Bortezomib treatment, including protein neddylation. The data also reveals a global increase in protein degradation, which suggests the induction of an autophagy-related process. The resulting data collected reveals significant changes upon Bortezomib treatment in protein synthesis of genes, including protein neddylation, and protein degradation data reveals a global increase in protein degradation, suggesting an induction of an autophagy-related process. Subsequent cellular and proteomic analysis investigated the additional treatment of an Autophagy inhibitor, hydroxychloroquine, in combination with Bortezomib treatment by label-free proteomics to further characterize the proteome-wide changes in these two proteotoxic stresses.

Conclusions: AHA metabolic labeling proteomics to investigate protein synthesis and degradation enables novel complementary insights into complex cellular responses compared to that of traditional label-free proteomics.

azidohomoalanine; proteasome; protein degradation; protein synthesis; proteomics; proteostasis.