Lon protease is a unique ATP-dependent protease found in all kingdoms of life [1]. In mitochondria, Lon represents a crucial protein responsible for maintaining the mitochondrial homeostasis and is important for degrading of misfolded, disassembled and oxidatively damaged proteins. Lon was also shown to control the levels of several mitochondrial proteins including the subunits of mitochondrial processing peptidase MPP, StAR protein, helicase Twinkle and ribosomal subunit MrpL32 in human cells, and mtDNA-packaging protein Abf2 and mtDNA-maintenance factor Mgm101 in S. cerevisiae [1-3]. To better understand the function of Lon, we determined the first 3D cryo-EM structure of a complete human mitochondrial Lon protease [4] and described the rearrangement of its structure when Lon is moving between the ATP-bound and ADP-bound states. Since both, over-expression and reduced expression of Lon, were found in cancer cells [5], it is clear that a more complex understanding of the mechanisms regulating its functions in mitochondria is needed. This regulation could occur on the level of its transcription, but recent studies have suggested that post-translational modifications might also have a large impact here. Phosphorylation represents an effective post-translational modification in cells. To characterize the effect of such modification on function and structure of human Lon protease, we prepared several point mutations in Lon′s N-terminal domain, where Tyr was replaced by Glu or, using an orthogonal translation system in modified E. coli strain, para-carboxymethyl-L-phenylalanine (pCMF), a non-hydrolysable analogue of phosphotyrosine, was introduced into the Tyr sites of interest. Our results indicated that phosphorylation of some N-terminal Tyr substantially reduced Lon protease activities without any significant change of its structure. Such regulation might facilitate quick dynamic changes to mitochondrial crucial components, nucleoids and ribosomes, which are inevitable for conducting mitochondrial functions and maintaining mitochondrial homeostasis in vivo and could also have significant implications for human medicine.
Acknowledgement The authors have received a support from the Slovak Research and Development Agency APVV-15-0375, APVV-19-0298), the Slovak Grant Agency (VEGA 2/0069/23), the Interreg V-A Slovakia-Austria program for the project StruBioMol, ITMS: 305011X666, co-financed by the European Regional Development Fund and Ministry of Education and CF CF CryoEM of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042)) and European Regional Development Fund-Project „UP CIISB“ (No. CZ.02.1.01/0.0/0.0/18_046/0015974).