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COX6B1 secures a redox-sensitive step in early cytochrome c oxidase assembly

ABSTRACT: COX6B is one of the eleven nuclear-encoded subunits of the human mitochondrial inner-membrane cytochrome c oxidase (cIV), which is located in the intermembrane space-facing region of cIV. In mammals, there are two COX6B isoforms: the ubiquitous isoform COX6B1, expressed in all cell types and tissues, and COX6B2 present only in testes and certain types of cancer. Rare COX6B1 missense pathogenic variants have been identified as the cause of childhood-onset mitochondrial encephalopathy associated with cIV deficiency. Despite the relevance of COX6B1 in mitochondrial physiopathology, its molecular role in cIV biogenesis and/or maintenance had not been thoroughly characterized. In spite of the assigned role for COX6B1 as a late incorporation subunit into an almost complete cIV, a COX6B1 human cell line knock-out (KO) exhibited a total loss of cIV. To get a deeper insight into the mechanisms that drive the lack of assembly or destabilization of cIV in the absence of COX6B1, we also used the COX6B1 KO cell background to express an alternative oxidase (AOX) and COX6B1 carrying the R20C and R20H pathogenic variants. These analyses have led us to conclude that the COX6B1 subunit does not only contribute to the stabilization of cIV in the late assembly stages, but is also indispensable for redox-sensitive early cIV assembly steps. In addition, this study has evidenced the incorporation of partially assembled cIV modules directly into supercomplex structures, supporting the Ã¢â‚¬Ëœcooperative assemblyÃ¢â‚¬â„¢ model for respiratory chain biogenesis.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Marek Vrbacky

LAB HEAD: Petr Pecina

PROVIDER: PXD061621 | Pride | 2025-12-15

REPOSITORIES: pride

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Project description:Ubash3b, also known as suppressor of T-cell receptor signaling or Sts-1, is an ill-studied atypical tyrosine phosphatase with ubiquitin binding ability. In our previous study, we hypothesized that Ubash3b plays an inhibitory role in BCR-ABL signaling through binding and dephosphorylating BCR-ABL and its interactors. The Hantschel lab recently solved the crystal structures of the p210 PH and DH domains, which are absent in the p190 variant, and demonstrated that loss-of-function mutations in the PH domain altered BCR-ABL localization, thereby reducing the interaction between Ubash3b and p2104. Taken together, this suggests differential subcellular localization of Ubash3b as a mechanism by which it interacts more strongly with p201 as compared to p190. To better understand the global impact Ubash3b has on p210, its direct kinase substrates and proteins in its phosphotyrosine signaling network, we have taken an integrative approach by combining global phosphotyrosine profiling, proximity-dependent biotinylation (BioID) and total protein analysis to investigate p210 signaling upon Ubash3b knockdown (KD). The BioID system was used to characterize Ubash3b function in p210 signaling by examining its interactome. Importantly, in all of our BioID experiments, we employed a newly technique that we have recently developed, Biotinylation Site Identification Technology (BioSITe), which directly identifies biotinylated peptides thereby increasing the reliability of the identified interactors. Here, we additionally used short hairpin RNA (shRNA) interference and generated Ubash3b knock-down (KD) and non-targeting control shRNA lines in Ba/F3 BirA*-p210 cells. Ubash3b expression was reduced >90 % in the KD cells and had a substantial effect on global tyrosine phosphorylation and on the interactome of p210. Of the 1,421 unique tyrosine phosphorylation sites identified from 830 proteins, 379 sites (from 286 proteins) exhibited a substantial increase (ÃƒÂ¢Ã¢â‚¬Â°Ã‚Â¥2-fold) in tyrosine phosphorylation upon Ubash3b KD cells compared to control cells. To date, the interactome of Ubash3b has not been extensively investigated, however, some examination of Ubash3b in the context p210 signaling has been undertaken. We designed constructs of C-terminal BirA* tagged full length Ubash3b and a deletion mutant lacking the UBA and SH3 domains leaving only the phosphatase domain tethered to BirA*. A comparative analysis of the core interactors of p210 from previous studies and Ubash3b interactome from the current study revealed 36 proteins that interact with both p210 and Ubash3b.

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COX6B1 secures a redox-sensitive step in early cytochrome c oxidase assembly

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