JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003767/files/210206_Koshiba_Nterm_mOMA1_FCCP_LFQ_FusionIT.xlsxhttps://storage.jpostdb.org/JPST003767/files/173_Koshiba_Nterm_1_KO_FusionIT.rawhttps://storage.jpostdb.org/JPST003767/files/177_Koshiba_Nterm_3_WT_FusionIT.rawhttps://storage.jpostdb.org/JPST003767/files/175_Koshiba_Nterm_2_KO_FCCP_FusionIT.rawhttps://storage.jpostdb.org/JPST003767/files/179_Koshiba_Nterm_4_WT_FCCP_FusionIT.rawprimary200OKProteomicsTakumi KoshibaMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST003767Tokushima 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 neo-amino terminal peptides using Tryp-NTo determine candidate OMA1 substrates during mitochondrial stress, OMA1 KO MEFs or its rescued cells expressing OMA1-Myc were treated with or without 40 uM FCCP for 2 h, then washed once with 1× PBS (pH 7.4), and cell pellets were collected. The pellets were then lysed in a guanidine buffer [6 M guanidine-HCl, 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP (Sigma Aldrich), and 40 mM 2-chloroacetamide (CAA, Sigma Aldrich)]. After heating and sonication, 30 ug of proteins was purified by methanol-chloroform precipitation and resuspended in 20 uL of PTS buffer [100 mM Tris-HCl (pH 8.0), 12 mM sodium deoxycholate, and 12 mM sodium lauroylsarcosinate]. The protein solution was diluted 10-fold with 10 mM CaCl2 and digested with 600 ng of Tryp-N (LysargiNase, Merck Millipore) at 37° C for overnight. After acidification with 0.5% TFA (final conc.), an equal volume of ethyl acetate was added to each sample, followed by centrifugation at 15,700g for 2 min to separate the ethyl acetate layer. The aqueous layer was collected and desalted using GL-Tip SDB, and the elutes were evaporated and dissolved in 50 uL of 2.5% formic acid and 30% acetonitrile. Enrichment of protein N-terminal peptides was performed using GL-Tip SCX (GL Sciences Inc.) based on the previous report (Chang et al, 2021). Flow-through fractions were evaporated and dissolved in 0.1% 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 for identification and label-free precursor ion quantification. Search parameters were as follows: (a) Tryp-N as a semi-specific enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) cysteine carbamidomethylation as a fixed modification; and (e) protein N-terminal acetylation and methionine oxidation as variable modifications.Sat Jan 31 00:00:00 GMT 2026PXD0630171009041876740