Effect of lysine post-translational modifications in the proteome of extracellular vesicles from non-small cell lung cancer cells

Exosomes, small cell-derived vesicles found in a wide range of biological fluids, play a pivotal role in numerous biological processes. They have been implicated in coagulation, protein degradation, immune signaling, antigen presentation, and cancer metastasis, underscoring their diverse and significant functions.

Emerging evidence suggests a potential link between ubiquitylation and protein export to exosomes, with acetylation potentially competing for the ε-amino groups of lysine. Given that enzymes tightly control the abundance of these modifications, it is plausible that the balance of these modifications, in concert with the metabolic state and microenvironment of the cells, governs the presence of specific proteins in exosomes. Importantly, dysregulation of acetylation and ubiquitylation, and the enzymes that govern them, has been implicated in the pathogenesis of non-small cell lung cancer.

Our hypothesis posits that the equilibrium of acetylation and ubiquitylation levels is a critical player in regulating protein export to EVs. This process is crucial in modifying vesicular cargo in various non-small cell lung cancer models.

We are using LC-MS/MS and the inhibition of deubiquitinases, proteasomal degradation, lysine acetyltransferases, and deacetylases to perform protein quantitation to evaluate and compare the changes in the profiles of intracellular proteins and the different populations of extracellular vesicles, characterizing the proteome and post-translational modifications (acetylation and ubiquitylation) in lysine residues (KPTM) in various NSCLC cell lines, and correlating these modifications to the targeting of proteins towards EVs, and comparing the protein composition of EVs in correlation to intracellular protein profiles in different non-small cell lung cancer models and blood-derived vesicles from cancer patients in different stages of the disease, with the possibility to identify tumor-related markers contained in EVs.

In the first stage of the analysis, we have identified 1638 proteins, highlighting proteins participating in vesicle formation, such as tetraspanins CD9, CD81 and CD151, the accessory protein ALIX (PDCD6IP), HGS protein (part of the ESCRT-0 complex), CHMB- 4b and IST1 (which is part of the ESCRT-III complex), and the GTPases Rab-7, Rab-35 and Rab-27, related to endosomal differentiation to multivesicular bodies, and their movement towards the plasma membrane to fuse and release exosomes contained in multivesicular bodies.

The results of this project will provide information on how the protein cargo of EVs is conformed during lung cancer development. Understanding this biological process can lead to the identification of biomarkers with diagnostic and prognostic value, and new therapeutic targets to stop metastasis formation.