title: Active-site-directed chemical tools for profiling mitochondrial Lon protease.
abstract: Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101-1709, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fishovitz
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.forename: Jason
authors.initials: J
authors.lastname: Hudak
authors.forename: Sandra
authors.initials: S
authors.lastname: Craig
authors.forename: Kristin
authors.initials: K
authors.lastname: Ko
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Berdis
authors.forename: Carolyn K
authors.initials: CK
authors.lastname: Suzuki
authors.forename: Irene
authors.initials: I
authors.lastname: Lee
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D049071:Endopeptidase Clp / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D049070:Protease La / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D010455:Peptides; D000255:Adenosine Triphosphate; D049070:Protease La; C568646:ClpP protein, human; D049071:Endopeptidase Clp; C528384:CLPX protein, human