Development and characterization of azido fatty acid protein modification in C2C12 myoblasts
Post-translational modification (PTM) of proteins adds immense complexity to the proteome. Roughly 90% of proteins are post translationally modified in some fashion, giving them new function, localization, or activity, which in turn changes their dynamic roles in intricate signal transduction cascades. It is key to understand and characterize the dynamic role that PTMs play in both normal and pathological cell functions to efficiently design pharmacological treatments for diseases in which PTMs are disregulated. In this work we focused on N-terminal myristoylation, a PTM that involves enzymatic addition of a fatty acid (myristic acid) onto the N-terminus of proteins. Methods were developed for incorporation of an azido fatty acid analogue of myristic acid (12-azidododecanoic acid - 12ADA) onto C2C12 myoblast proteins. Detection and analysis were carried out utilizing copper-catalyzed click chemistry. 12ADA labeled C2C12 lysates were reacted with fluorescent probes and analyzed via SDS-PAGE, allowing identification of proteins labeled via myristoylation relative to the entire proteome of C2C12 lysate. Fixed C2C12 cells were like-wise reacted with fluorescent probes and imaged. Results displayed localization of distinct puncta of myristoylated proteins within the cell as well as varying 12ADA incorporation levels in differentiated C2C12 cells versus myoblastic cells. The study of the dynamics of protein myristoylation was enabled with time-course studies of 12ADA incorporation. Findings from this work characterize a complex system of myristoylation in C2C12 cells. The developed techniques are easily translatable as tools for deciphering the function of myristoylation in varying cell physiological states in this or other cell lines and for other post-translational modifications, such as palmitoylation, within cellular protein signaling networks.
Kinzer-Ursem, Purdue University.
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