Neprilysin protease substrates: degradomics reveals novel roles in mitochondria and in neurodegeneration

The role of proteases in neurodegeneration is central and extensively investigated. Recently, by using the mitochondrial dimethylation-TAILS degradomics approach in a cellular model of dopamine (DA) imbalance in neuroblastoma SH-SY5Y cells, we demonstrated that Neprilysin is a candidate protease involved in mitochondrial dysfunction related to Parkinson"s disease (PD)¹. Neprilysin is a Zinc-dependent endopeptidase, which exists in two catalytically active forms, i.e., transmembrane, and soluble. It is a recognized biotarget, being responsible for the cleavage of biologically relevant substrates such as angiotensin, bradykinin, substance P, glucagon, insulin β-chain, oxytocin, enkephalins, and amyloid-beta.

To investigate the role of neprilysin as a candidate mitochondria-localized protease, we first verified its presence and analysed its localization inside mitochondria, by sub-fractionation experiments and fluorescence staining. Moreover, we observed a markedly increased co-localization between neprilysin and mitochondria upon the induction of DA imbalance in SH-SY5Y cells. Then, to obtain the complete set of neprilysin substrates, we applied the dimethylation-TAILS approach on the total proteome of SH-SY5Y cells. A list of 155 candidate substrates emerged, and twenty of them resulted to be mitochondria-associated proteins, including some ribosomal proteins, heat shock proteins, membrane transporters, and metabolic enzymes. The functional enrichment analysis highlighted "protein translation", "ribosomal function", "processing of RNAs and proteins", and "neurodegeneration" as significantly enriched pathways in the whole substrates list. In conclusion, we demonstrated for the first time that neprilysin exists as a mitochondrial protease and plays a role in mitochondrial dysfunction linked to DA imbalance in PD. These processes are central to understand early events in PD development.

¹ Lualdi, M.; Ronci, M.; Zilocchi, M.; Corno, F.; Turilli, E. S.; Sponchiado, M.; Aceto, A.; Alberio, T.; Fasano, M. Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson"s Disease. Front. Aging Neurosci. 2019, 11, 195. https://doi.org/10.3389/fnagi.2019.00195.