Fig 1: The L280F variant in BCS1L, identified in two siblings with a neuromuscular disorder and axonal motor neuropathy, exhibits impaired binding to the Rieske protein. (A) Schematic of the molecular pathway involved in Fe‐S cluster delivery to UQCRFS1, followed by its insertion into the CIII precomplex. Binding of the cochaperone HSC20 to the accessory factor LYRM7 facilitates Fe‐S cluster incorporation into UQCRFS1 [10] in an ATP‐dependent process that might involve intermediary carriers. [2Fe‐2S]‐UQCRFS1 is subsequently inserted into the CIII precomplex by BCS1L, and CIII assembly is completed by the acquisition of the last subunit QCR10. (B) Heptameric ensemble of Mus musculus Bcs1l (PDB ID: 6UKO) with L280 highlighted. (C) Multiple sequence alignment of BCS1L sequences reveal complete conservation of the L280 residue across species, with the exception of Anopheles gambiae , which has an isoleucine at this position. (D,E) Structural analysis of monomeric Bcs1l reveals the proximity of L280 to E282, a Walker B motif residue crucial for ATP hydrolysis (D). The L280F substitution results in steric hindrance between F280 and the catalytic residue E282 (E). (F) Co‐IP experiments in HeLa cells comparing BCS1L wild‐type and L280F variant. The L280F variant shows impaired recruitment of the Rieske protein. Input samples (20%) and unbound lysates after DDK‐bead incubation are included for comparison. (unb. = unbound). (G) Co‐IP experiments in HeLa cells expressing wild‐type BCS1L and its variants L280F, S78G, and R183H. Cells transfected with the empty vector (pCMV6‐Entry) served as a negative control. Immunoblotting (IB) was performed using antibodies against BCS1L to detect both endogenous and recombinant proteins, and against DDK (FLAG) for recombinant BCS1L only. IB against LYRM7 was conducted to probe for a potential BCS1L‐UQCRFS1‐LYRM7 complex, which was not detected. This finding is consistent with the current understanding that UQCRFS1 incorporation into the CIII assembly intermediate occurs after its Fe‐S cluster acquisition [10, 12]. (F and G, n = 4 biological replicates).
Fig 2: Patient‐derived cells exhibit decreased OCR and elevated markers of oxidative stress compared to paternal cells. (A) Normalized oxygen consumption rates (OCR) in patient‐derived, paternal, and control (FC2) cells. FC2 cells are fibroblasts from an individual carrying two BCS1L wild‐type alleles. (B) Basal OCR in patient‐derived and control (paternal and FC2) cells (n = 8 biological replicates). (C) Spare respiratory capacity in patient‐derived and control (paternal and FC2) cells (n = 8 biological replicates). (D) Markers of oxidative damage [4‐hydroxynonenal (4‐HNE), malondialdehyde (MDA), and protein carbonyl content] in patient‐derived and control (FC2 and paternal) cells (n = 9 biological replicates). (E) ROS levels in patient‐derived and control (FC2 and paternal) cells as assessed by flow cytometry (FACS, n = 9 biological replicates). (**p < 0.01; ***p < 0.001; ****p < 0.0001).
Fig 3: Patient‐derived cell lines demonstrate reduced BCS1L protein levels, defective CIII assembly, and decreased enzymatic activity compared to paternal cells. (A) Protein levels of mitochondrial components in patient‐derived (P1 and P2) and parental (father) cells. Immunoblotting (IB) was performed to detect BCS1L, LYRM7, UQCRFS1, NDUFS1 (complex I Fe‐S subunit), and MTCO1 (complex IV heme and copper subunit). TOM20 served as a mitochondrial loading control. (B) Quantification of immunoblots (n = 4) equivalent to those presented in panel A performed using Image Lab Software (Bio‐Rad). Data points represent individual measurements. Error bars indicate standard deviation (SD). (C) CIII activity assay in patient‐derived (P1 and P2), paternal and control (CTRL) fibroblast cells. CTRL represents a fibroblast cell line carrying two wild‐type copies of BCS1L. (D) Native IBs against UQCRC1, MTCO1, and NDUFS1 to detect levels of fully assembled complexes III, IV, and I, respectively, and in‐gel NADH dehydrogenase activity of CI in patient‐derived and paternal cells. A‐C, n = 4 biological replicates. (****, p < 0.0001) (E) Iron content in cytosolic (CYT) and mitochondrial (MIT) fractions isolated from patient‐derived (P1 and P2), paternal, and control cells as measured by ICP‐MS. No statistically significant differences were observed between experimental groups (n = 4 biological replicates). (F) Labile iron pool (LIP) in patient‐derived (P1 and P2), paternal, and control cells. P1 exhibited significantly elevated LIP compared to P2, paternal, and control cells. P2 LIP levels were not statistically different from paternal or control cells (n = 6 biological replicates).
Fig 4: Identification of biallelic variants in BSC1L. (A) Pedigree, showing autosomal recessive inheritance with shared biallelic BCS1L variants in affected siblings. Both parents are each heterozygous carrier for one of the pathogenic BCS1L variants. The two affected siblings (P1 and P2) are compound heterozygous for the biallelic BCS1L variants‐ each inherited from the respective carrier parent. WT = wild type. (B) Genomic and protein domain representations of isoform NM_001079866 of BCS1L. Patient variants are indicated in the 5′‐UTR region and exon 6. (C,D) Panels are adapted from the UCSC Genome Browser. (C) The 5′‐UTR maternally inherited variant is mapped upstream to exon 1 and highlighted in light blue. Regulatory tracks: ENCODE Registry of candidate cis‐Regulatory Elements (cCREs) and enhancers and promoters from GeneHancer show that the variant falls within a strong regulatory region. The green and blue tracks highlight robust evolutionary conservation in 30 mammalian species. (D) The UCSC 100 Vertebrates track displays multiple sequence alignments across vertebrate species and assessment of evolutionary conservation of leucine in position 280.
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