JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003766/files/201222_Koshiba_mOMA1_Myc_IP_n3_FusionIT.xlsxhttps://storage.jpostdb.org/JPST003766/files/151_Koshiba_5_OMA1_WT2_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/155_Koshiba_3_OMA1_KO3_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/143_Koshiba_1_OMA1_KO1_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/157_Koshiba_6_OMA1_WT3_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/147_Koshiba_7_OMA1_EQ1_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/149_Koshiba_2_OMA1_KO2_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/159_Koshiba_9_OMA1_EQ3_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/145_Koshiba_4_OMA1_WT1_FusionIT.rawhttps://storage.jpostdb.org/JPST003766/files/153_Koshiba_8_OMA1_EQ2_FusionIT.rawprimaryOK200ProteomicsTakumi KoshibaMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST003766Tokushima UniversityjPOSTMitochondrial proteases regulate dynamic properties of organelle morphology and ensure functional plasticity at the cellular level. The metalloprotease OMA1 mediates constitutive and stress-inducible processing of its mitochondrial substrates, although only a few of its direct functional targets have been characterized. Using in vitro and in vivo multiproteomic and biochemical approaches, we here demonstrate that the membrane-anchored intermembrane space (IMS) protein AIFM1 serves as a mitochondrial stress-responsive OMA1 substrate. Under stress conditions, OMA1 cleaves AIFM1 in the IMS with slower kinetics than its conventional substrate, the dynamin-like GTPase OPA1. OMA1-mediated dislocation of cleaved AIFM1 from the mitochondrial inner membrane reduces its interaction with oxidative phosphorylation subunits, thereby decreasing respiratory activity and impairing cell growth. Furthermore, we reveal that under steady-state conditions AIFM1 broadly safeguards the mitochondrial proteome by mediating the import of proteins, particularly respiratory complex I subunits, via the TIM23 complex. Similar changes to the mitochondrial proteome occur in the lungs of virally infected mice, accompanied by stress-inducible AIFM1 processing. These findings identify OMA1 as a key integrator of mitochondrial stress and cellular energetics through AIFM1 remodeling.Stress-induced OMA1-mediated cleavage of AIFM1 suppresses cell growth by controlling mitochondrial OXPHOS activity.Nishigori Mitsuhiro M, Hirata Serina S, Kosako Hidetaka H, Ichinohe Takeshi T, Nolte Hendrik H, Riemer Jan J, Langer Thomas T, Koshiba Takumi TfalseLFQ of anti-Myc IP-MS of OMA1-Myc-expressing MEFsOMA1 KO MEFs (10 cm dish) and its rescued cells stably expressing WT or EQ mutant OMA1-Myc were incubated with 0.2% (w/v) formaldehyde for 15 min at 37° C, followed by quenching with 100 mM glycine-NaOH (pH 7.5) for 10 min at room temperature. After once wash with 1× PBS (pH 7.4), the cells were scraped and mitochondria were isolated from the cells as described above. The mitochondrial fractions were then lysed in 1 mL of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% (w/v) glycerol, 1 mM EDTA, 0.5% (w/v) digitonin, and protease inhibitor cocktail. After centrifugation, the clarified supernatants were incubated with an antibody against Myc (My3; MBL Life Science, Tokyo, Japan) for 2 h at 4° C. The reactants were then incubated with magnetic SureBeads Protein G (Bio-Rad) overnight at 4° C, and the next day the beads were washed three times with 1× PBS (pH 7.4) and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) for 16 h at 37° C. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB, and the eluates were evaporated and dissolved in 0.1% trifluoroacetic acid (TFA) and 3% acetonitrile. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. Peptides were separated on a 75 µm inner diameter × 150 mm C18 reverse phase column (Nikkyo Technos, Tokyo, Japan) with a linear gradient of 4%-32% acetonitrile for 0-100 min followed by an increase to 80% acetonitrile for 100-110 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were analyzed directly against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) for identification and label-free precursor ion quantification. Search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; and (d) cysteine carbamidomethylation as a fixed modification; and (e) protein N-terminal acetylation and methionine oxidation as variable modifications. Peptides were filtered with a false discovery rate of 1% using the percolator node. Normalization was performed so that the total sum of abundance values for each sample was equal across all peptides.Sat Jan 31 00:00:00 GMT 2026PXD0630141009041876740