Identification of ADAM17 SUMOylation, examination of its effect on protein stability, and confirmation of Cas9 post-translational modifications by proximity ligation assays

Post-translational modifications, such as SUMOylation and ubiquitylation, are essential regulatory mechanisms that modulate various cellular processes, including gene expression, protein function, and signaling. These modifications can alter the stability, localization, and interactions of target proteins, thereby regulating a broad range of biological activities. This study investigated the roles of SUMOylation and ubiquitylation on CRISPR-associated Cas9 and the metalloprotease ADAM17. Understanding the regulation of Cas9 through post-translational modifications is critical for improving the efficiency and specificity of CRISPR-based genome editing. In this study, a powerful and ultra-sensitive technique called proximity ligation assay (PLA) was employed to further verify that Cas9 is both SUMOylated and ubiquitylated. The PLA experiments also confirmed the major SUMO conjugation site of Cas9. Furthermore, PLAs have shown that Cas9's SUMOylation and ubiquitination modulate its subcellular localization by competing for the same lysine residue and acting antagonistically. The regulation of ADAM17 activity by post-translational modifications is a critical area of research, as ADAM17 is a key enzyme that cleaves and releases numerous cell surface proteins, including cytokines, growth factors, and their receptors. This study showed for the first time that ADAM17 is subject to both SUMOylation and ubiquitylation and that these modifications have opposing effects on ADAM17 stability. However, further research is needed to identify the specific SUMOylation sites and their impact on ADAM17 stability, localization, and shedding activities.