Unveiling hidden protein dynamics: a multi-proteomic approach to study new players in metastatic melanoma progression

In our recent study, we uncovered amyloid-related proteins and fibrillar aggregates in the extracellular environment of metastatic melanoma cells. Intriguingly, there is only a known physiological amyloidogenic process in humans occurring in melanocytes, where BACE2 processes PMEL protein into amyloidogenic peptides to create a melanin scaffold. This process seems deregulated in metastatic melanoma, leading to aberrant fibril deposition. BACE2 is upregulated in many tumors, and our findings showed that inhibiting its activity in metastatic melanoma cells decreased cell proliferation and aggregate deposition.The Limited Proteolysis coupled to Mass Spectrometry (LiP-MS) approach was employed to analyze the metastatic melanoma secretome following BACE2 inhibition. This approach permitted the study of protein aggregation in the biological environment, highlighting conformational perturbations. BACE2 is a sheddase located both intracellularly and at the plasma membrane and regulates many proteolytic processes, potentially causing aberrant shedding of many transmembrane proteins. The N-Tails degradomics approach assessed the global proteolytic process mediated by BACE2. LiP-MS secretome analysis revealed a conformational perturbation in four known amyloidogenic proteins, including PMEL. Conformational rearranged peptides were found in two domains within the amyloidogenic core, and N-Tails showed cleavages close to these domains. This suggested that amyloidogenic secreted proteins were in a fibrillar conformation. Interestingly, recombinant PMEL amyloid fibrils added to the extracellular environment promoted migration and invasion in metastatic melanoma cells, indicating that deregulated fibrillation supports tumor progression. Through N-Tails, we observed other transmembrane proteins affected by BACE2-related cleavage, and we are now characterizing these targets in relation to their activity in metastatic melanoma. These findings demonstrate that BACE2 significantly rearranges the extracellular environment by influencing the secretion of pro-tumoral fibrils. Moreover, these data open up the possibility of decoding strategies employed by the tumor to regulate cellular processes through BACE2 cleavage. Using a multi-proteomic approach, we gained insights into post-translational and conformational modifications of proteins, beyond the abundance perturbations detected by shotgun proteomics, thus facilitating our understanding of underlying biological mechanisms.