{"database":"iProX","file_versions":[],"scores":null,"additional":{"omics_type":["Proteomics"],"submitter":["Hengbin Wang"],"species":["Homo Sapiens"],"full_dataset_link":["http://www.iprox.org/page/project.html?id=IPX0012515000"],"submitter_email":["hengbin.wang@vcuhealth.org"],"submitter_affiliation":["Department of Internal Medicine, Division of Hematology, Oncology and Palliative Care, Massey Comprehensive Cancer Center"],"sample_protocol":[""],"repository":["iProX"],"data_protocol":[""],"name_synonyms":["W53272, SPBC8D2.22, PEREC1, USP16, Mitochondrial, outer mitochondrial membrane, D19S1177E, PER-EC1, l(1)G0216, ISP42, Usp23, dtom40, AW539759, translocase of the outer mitochondrial membrane 40, function, tom40, mit, ESTM28, 2810483I07Rik, mitochondrial, CG12157, mitochondrion outer membrane., USP23, DmelCG12157, ATP synthase D chain, TOM40, UBPM, UBP-M, C19orf1, ms(1)15, core, MOM38, Usp16, 6330514E22Rik, 1200004E02Rik, ms15, outer mitochondrion membrane, Mom35, Tom40"],"description_synonyms":["Panomics, tub, Co-Immunoprecipitations, Cognitive Function, Reverse, Immunoelectroblotting, Fs(3)Kavar, alphatub67C, C20orf14, outer mitochondrial membrane, alpha-Tub67C, determination, Lysine Hydrochloride, Antibody Technics, Membrane Potential, Inner, hPrp6, ANT-1, Bbu, sci, Technic, Mitochondrial Contractions, Co Immunoprecipitation, Histone H2b, Histone H2a, Techniques, Mitochondrial Membranes, Lysine Acetate, Electroimmunoblotting, alphaTub[67C], Immunofluorescence Technics, Roles, symptoms, HOW, How, Concepts, 2, barbu, Transmembrane Potential Differences, establishment and maintenance of substrate location, Pro Oxidant, l(3)j5D5, IKKg, KEY, Key, mitochondrion outer membrane, L Lysine, W53272, 24B, DmelCG8308, ubiquitin hydrolase activity, pore, High Mobility Protein 20, Protein Tracings, Coon Technic, Immunoblottings, K, alphaTub4, Differences, Co-Immunoprecipitation, dtom40, stru, AW539759, Precipitation, epsilon-diaminocaproic acid, T, Ubiquitin-related 1, D.m.ALPHA-67C, l(3)S053606, mit, ESTM28, ubiquitin C-terminal hydrolase, CG10293, Delta Psi, l(3)j5B5, APF-1, Oxygen, Cognitions, Immune, LYS, Inner Mitochondrial Membrane, Functions, Lysin, Role Concepts, Transmembrane Potentials, Histone H5, Histone H4, Histone H7, Precipitations, outer mitochondrion membrane, ms15, alpha-Tub, Histone H1, Histone H3, ubiquitin C-terminal hydrolase activity, Deubiquitinating, DTA2, establishment and maintenance of substance location, Fluorescent Antibody Techniques, screening, 0904/17, alpha4-t, Fluorescent Antibody, Difference, Resting Potential, l(1)G0216, Oxygen Radical, U5-102K, Fluorescent, Insight, alpha-tubulin, CEP52, Fs(3)Sz14, single organism localization, DmIKKgamma, Role Concept, ubiquitin-specific protease 16, ATP synthase D chain, positive regulation of homeostatic process, alpha-tubulin[4], SZ1, dIKK, CG5185, UBP-M, Inner Mitochondrial Membranes, Coons Technic, Kenny, Role, lysine, tub67c, Technics, Antibody Techniques, regulator, Oxygen Radicals, Ubiq, Fs(3)Sz22, Reverse Immunoblottings, 4t, Ubiquitin, Protein Tracing, Multi Omics, Mitochondrial, Potential Differences, D19S1177E, function., PER-EC1, Outer, Deubiquitinases, Radical, Consumptions, IKK-gamma, Integrative, Human Ubiquitin, signs, alpha, 2810483I07Rik, CG12157, DmelCG16910, Coon's Technique, Reverse Immunoblotting, Fluorescent Antinuclear Antibody Test, Resting Potentials, Cells, Fluorescent Protein Tracings, C19orf1, CG8308, ms(1)15, Fluorescent Protein Tracing, MOM38, anon-EST:Liang-2.39, aTub67C, 6330514E22Rik, Resting Membrane Potentials, Mom35, establishment and maintenance of cellular component location, Fs(3)Tom, protein levels, Outer Mitochondrial, Transmembrane, ATP regeneration, Immunoblotting, Outer Mitochondrial Membranes, Potential, P62, Fluorescent Protein, ubiquitinyl hydrolase 1 activity, Dot Immunoblotting, Reactive Oxygen Intermediates, translocase of the outer mitochondrial membrane 40, ATP Dependent Proteolysis Factor 1, Deubiquitinating Enzyme, Immunofluorescence Technic, Ubiquitin carboxyl extension protein 80, Potential Difference, mitochondrial, Mitochondrial Membrane, deubiquitylase, dIKK-gamma, Human, Multi-Omics, deubiquitinase activity, Dot, DmIKK-gamma, Mitochondrion, establishment and maintenance of position, Immune Precipitations, Tom, TOM, HMG-20, Lysine, dmIKKgamma, IKK[[gamma]], alpha4, Technique, Tracing, Immune Precipitation, Reactive, Histone H3.3, PEREC1, study, alpha-tub67C, l(3)s2612, Transmembrane Electrical Potential Difference, Transmembrane Potential, Contraction, Prp6, ISP42, HeLa, tom, ANT1, Omics, Oxygen Species, RP60, Cognitive Functions, UBP, 40S ribosomal protein S27a, Enzyme, IKK, Active Oxygen Species, ALPHA 67C, UBPM, ubiquitin, DmelCG10293, negative regulation of homeostatic process, Coon's, Pan-Omics, Immunoelectroblottings, Pro-Oxidant, Immunofluorescence Techniques, protein tagging activity, Active Oxygen, alpha4t, mum-tub67C, Oxygen Consumptions, SNRNP102, SPBC8D2.22, Autoregulation, Tracings, F22K18.240, Integrative-Omics, Pro Oxidants, findings, Coon's Technic, clone 2.39, Fs(3)Kav, alpha4-tubulin, Deubiquitinase, function, Fluorescent Antibody Technics, qkr, Integrative Omics, alphatub, l(3)S090417, Histone, Fluorescent Antibody Technic, Cell, bbu/tom, alpha-tub, Concept, Fs(3)Tomaj, Consumption, Enzymes, Contractions, IKKgamma, Insights, Ubiquitin A-52 residue ribosomal protein fusion product 1, Dot Immunoblottings, HeLa Cell, Ubiquitin-related 2, Acetate, KH93F, chemical analysis, Antinuclear Antibody Test, Multi-Omic, Pan Omics, core, Active, establishment and maintenance of localization, Immunofluorescence Technique, ATP-Dependent Proteolysis Factor 1, Antibody Technique, 1200004E02Rik, regulation of homeostatic process, L-Lysine, alphaTUB, alphaTub, who, Kavar, Membranes, covalent modifier, USP16, Electroimmunoblottings, Coons Technique, Dmikkgamma, Resting, Usp23, Function, pore-forming toxin activity, Who/How, single-organism localization, tom40, Membrane, CG16910, Cognitive, Coon Technique, Histone H1(s), UCH2, Mitochondrial Contraction, Outer Mitochondrial Membrane, Tub, anon-EST:fe1F6, F22K18_240, deubiquitinating enzyme, Transmembrane Potential Difference, mitochondria, Antibody Technic, USP23, localisation, DmelCG12157, 60S ribosomal protein L40, TOM40, Inner Mitochondrial, deubiquitinase, Pro-Oxidants, qkr[93F], alpha4Tub67C, Ubiquitin-related, Potentials, Resting Membrane Potential, Usp16, Membrane Potentials, assay, 6-diaminohexanoic acid, Enisyl, Immunofluorescence, alpha67C, Tom40, channel-forming toxin activity, DmelCG5185"],"additional_accession":[]},"is_claimable":false,"name":"USP16 Regulates Mitochondrial Function by Deubiquitinating TOM40, A Core Component of the Translocase in Mitochondrial Outer Membrane_AP-MS","description":"The ubiquitin-specific protease 16 (USP16), initially identified as a histone H2A-specific deubiquitinase. However, whether endogenous USP16 localizes to mitochondria and, if so, how it regulates mitochondrial function remain unclear. Therefore, this study focused on USP16, systematically investigating its localization, function, and regulatory mechanisms in mitochondria through various experimental approaches. Methods： First, we systematically investigated the subcellular localization of USP16 using immunofluorescence (IF), subcellular fractionation, and immunoblotting analysis. To further explore the function of USP16 in mitochondria, we conducted USP16 rescue experiments in USP16 knockout (KO) HeLa cells and performed mitochondrial function assays. To uncover the specific mechanism of USP16 in mitochondria, we successfully identified the key substrate of USP16—the translocase of outer mitochondrial membrane 40 (TOM40)—using multi-omics mass spectrometry analysis, co-immunoprecipitation, and immunofluorescence experiments. To further validate the role of TOM40 in USP16-regulated mitochondrial function, we overexpressed TOM40 in USP16 KO cells and then performed mitochondrial functional assays to confirm whether TOM40 is the critical downstream target through which USP16 regulates mitochondrial function and maintains metabolic homeostasis. Results： We demonstrate that USP16 interacts with and regulate the stability TOM40, the core pore-forming subunit of the translocase complex in the outer mitochondrial membrane (TOM). Specifically, USP16 deubiquitinates K48-linked ubiquitin chains from lysine 175, 184, and 309 of TOM40. Knockout of USP16 disrupts mitochondrial homeostasis, resulting in impaired membrane potential, elevates reactive oxygen species, reduces ATP synthesis, and alters oxygen consumption rates. Expression of FLAG-tagged USP16 restores TOM40 protein levels and rescues mitochondrial function. The essential role of TOM40 in mitochondrial homeostasis is further supported by partial restoration of mitochondrial functions upon TOM40 re-expression in USP16 KO cells. Conclusion: These findings identify USP16 as a novel regulator of mitochondrial function and establish TOM40 as an essential target of USP16 in mitochondria, providing valuable insights into the mechanisms by which USP16, acting as a dual-function deubiquitinase, supports mitochondrial integrity and function.","dates":{"publication":"Sat Jul 05 00:00:00 BST 2025"},"accession":"PXD065823","cross_references":{"TAXONOMY":["9606"]}}