title: Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.
abstract: Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.
authors.affiliation: Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and.
authors.forename: Lara
authors.initials: L
authors.lastname: Gibellini
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;
authors.forename: Marcello
authors.initials: M
authors.lastname: Pinti
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;
authors.forename: Federica
authors.initials: F
authors.lastname: Boraldi
authors.affiliation: Department of Biomedical Sciences, University of Padova, Padua, Italy;
authors.forename: Valentina
authors.initials: V
authors.lastname: Giorgio
authors.affiliation: Department of Biomedical Sciences, University of Padova, Padua, Italy;
authors.forename: Paolo
authors.initials: P
authors.lastname: Bernardi
authors.affiliation: Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and.
authors.forename: Regina
authors.initials: R
authors.lastname: Bartolomeo
authors.affiliation: Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and.
authors.forename: Milena
authors.initials: M
authors.lastname: Nasi
authors.affiliation: Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and.
authors.forename: Sara
authors.initials: S
authors.lastname: De Biasi
authors.affiliation: Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara, Ferrara, Italy;
authors.forename: Sonia
authors.initials: S
authors.lastname: Missiroli
authors.affiliation: Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Carnevale
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;
authors.forename: Lorena
authors.initials: L
authors.lastname: Losi
authors.affiliation: Biosciences Laboratory, IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Italy;
authors.forename: Anna
authors.initials: A
authors.lastname: Tesei
authors.affiliation: Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara, Ferrara, Italy;
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;
authors.forename: Daniela
authors.initials: D
authors.lastname: Quaglino
authors.affiliation: Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and Dipartimento Sperimentale Interaziendale, Campus San Lazzaro, University of Modena and Reggio Emilia, Reggio Emilia, Italy andrea.cossarizza@unimore.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cossarizza
mesh_terms: D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D015536:Down-Regulation; D020868:Gene Silencing; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D009369:Neoplasms / Q000201:enzymology* / Q000473:pathology; D049070:Protease La / Q000235:genetics*; D020543:Proteome; D034622:RNA Interference; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome; D049070:Protease La
keywords: RKO cells; mtDNA; mtRNA; oxphos; respiration