SEARCHOTHERmitochondrial medicine, neurology

other·mitochondrial medicine, neurology, genetics, metabolism, pediatrics·PMC10087869

Loss of mitochondrial fatty acid β‐oxidation protein short‐chain Enoyl‐CoA hydratase disrupts oxidative phosphorylation protein complex stability and function

The Febs Journal · 10 authors, 10 centres

AI SUMMARY

POPULATIONCRISPR/Cas9 ECHS1 knockout 143BTK− osteosarcoma cells and fibroblasts from nine patients diagnosed with ECHS1 deficiency (ECHS1D)

INTERVENTIONLoss of ECHS1 expression (knockout or pathogenic mutations)

COMPARISONControl 143BTK− cells (CON) and fibroblasts from healthy subjects

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TL;DR

This study demonstrates that loss of the mitochondrial fatty acid β-oxidation protein ECHS1 disrupts oxidative phosphorylation (OXPHOS) complex stability and function, particularly complex I and IV. Using CRISPR/Cas9 ECHS1 knockout cells and fibroblasts from nine ECHS1 deficiency (ECHS1D) patients, the authors show reduced OXPHOS complex steady-state levels, decreased enzymatic activity, and impaired respiratory capacity. These findings establish that secondary OXPHOS dysfunction contributes to the pathogenesis of ECHS1D, a subtype of Leigh Syndrome, and suggest ECHS1 interacts with complex I subunits NDUFB11 and NDUFV3 to support complex I assembly.

Full summary

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**Background:** ECHS1 (short-chain enoyl-CoA hydratase 1) catalyzes the second step of mitochondrial fatty acid β-oxidation (FAO) and is also involved in branched-chain amino acid breakdown. ECHS1 deficiency (ECHS1D) is a specific subset of Leigh Syndrome (LS), a neurodegenerative disorder associated with over 75 genes. Since 2014, more than 46 cases with confirmed ECHS1 mutations have been reported. Patients present with developmental delay, dystonia, cardiomyopathy, brain lesions, and metabolic acidosis, with a median lifespan of 2 years. Many ECHS1D patients exhibit reduced activity of OXPHOS complex enzymes, suggesting secondary OXPHOS defects, but the underlying mechanism was unknown.

**Methods:** The authors generated an ECHS1 'knockout' (KO) cell line using CRISPR/Cas9 gene editing in 143BTK− osteosarcoma cells, confirmed by Sanger sequencing (compound heterozygous mutation: 11-bp deletion p.Lys92GlyfsX40 and 44-bp deletion with 21-bp insertion p.Iso85LeufsX34) and absence of ECHS1 protein by western blot. RNA-seq was performed on ECHS1 KO and control cells (n=4). Proteomics was conducted on isolated mitochondria via LC-MS/MS. OXPHOS complex steady-state levels were assessed by SDS/PAGE and BN-PAGE western blotting. OXPHOS enzyme activities (complex I–IV) were measured spectrophotometrically. Mitochondrial respiration was measured using high-resolution respirometry in intact cells (glucose or palmitoyl-L-carnitine) and isolated mitochondria (complex I- or II-linked substrates). ROS production was assessed using H2DCFDA fluorescence. Co-immunoprecipitation (Co-IP) with anti-ECHS1 antibodies followed by mass spectrometry identified putative ECHS1-interacting proteins. Radiolabeled NDUFA9 import/assembly assays assessed complex I biogenesis. Fibroblasts from nine ECHS1D patients were analyzed for OXPHOS protein steady-state levels.

**Key Results:** RNA-seq revealed 11,278 differentially expressed genes (FDR<0.05) in ECHS1 KO cells (5,359 upregulated, 5,919 downregulated). Multiple OXPHOS transcripts were downregulated, including subunits of complex I (e.g., NDUFB11 −0.30 log2 fold change, NDUFV3 −0.30), complex II (SDHA −0.48), complex III (UQCRC2 −0.76), and complex IV (COX5B −0.22, COX7B −0.27, COX8A −0.21, COX10 −0.66, COX18 −0.43, COX20 −0.55). All six complex I assembly modules had reduced subunit or assembly factor expression. Proteomics identified 274 significantly altered mitochondrial proteins (142 upregulated, 132 downregulated; FDR<1%). Downregulated pathways included fatty acid β-oxidation, oxidoreductase activity, and aldehyde dehydrogenase activity. SDS/PAGE western blotting showed reduced steady-state levels of NDUFB8 (49.8±8.1%, P<0.01) and MT-CO2 (64.0±9.8%, P<0.05) in ECHS1 KO cells. BN-PAGE revealed decreased levels of mature complex I (69.4±10.4%, P<0.05), complex III dimer (41.8±20.1%, P<0.05), complex IV (47.5±1.4%, P<0.001), complex V (62.4±1.4%, P<0.01), CI/CIII2/CIV supercomplex (55.7±16.4%, P<0.05), and CIII2/CIV supercomplex (22.2±8.7%, P<0.01). Complex I activity was reduced to 42.6±39.9% (P<0.05) when normalized to citrate synthase, and complex IV activity to 66.3±9.4% (P<0.05). Raw enzymatic rates showed complex I at 21.4±6.6% (P<0.01) and complex IV at 28.2±4.2% (P<0.001) of controls. Citrate synthase activity was also reduced (31.1±4.6%, P<0.0001). Intact ECHS1 KO cells showed reduced basal (69.2±4.4%, P<0.05) and maximal (57.5±1.8%, P<0.05) respiration with glucose, and more severely reduced respiration with palmitoyl-L-carnitine (basal 26.8±12.3%, P<0.05; maximal 42.8±16.5%, P<0.05). Isolated mitochondria showed reduced state IV respiration with complex I-linked substrates glutamate/malate (71.5±21.4%, P<0.01) and pyruvate/malate (76.9±4.4%, P<0.05), but not with succinate. ECHS1 KO cells were insensitive to rotenone inhibition of ROS production. Co-IP identified six putative ECHS1-interacting proteins: NDUFB11, NDUFV3, ATP5F1D, ACADVL, RPL12, and RPL29. Radiolabeled NDUFA9 assembly into mature complex I after 60 min was reduced to 37.9±19.4% (P<0.01) of control levels in ECHS1 KO cells. In ECHS1D patient fibroblasts, ECHS1 was undetectable in 7/9 patients. NDUFB8 was decreased in 6/9 patients (38–52% of control), UQCRC2 decreased in 5/9 (39–53%), MT-CO2 decreased in 5/9 (~40%), and ATP5A decreased in 2/9 (~50%). Native complex III dimer was reduced in 6/9 patients (~30% of control), complex IV in 3/9 (~50%), complex V in 2/9 (~25%), and CI/CIII2/CIV supercomplex in 3/9 (~35%).

**Clinical Implications:** This study provides mechanistic evidence that loss of ECHS1 causes secondary OXPHOS dysfunction through disrupted complex I assembly, likely via lost interactions with NDUFB11 and NDUFV3, leading to reduced complex I and IV stability and activity. These findings explain the combined FAO and OXPHOS defects observed in ECHS1D patients and establish OXPHOS dysfunction as a key contributor to disease pathogenesis. The results suggest that therapeutic strategies targeting OXPHOS function, particularly complex I, may be relevant for ECHS1D. The study also highlights that ECHS1D should be considered in the differential diagnosis of Leigh Syndrome with combined FAO and OXPHOS defects.

PICO

PPOPULATION

CRISPR/Cas9 ECHS1 knockout 143BTK− osteosarcoma cells and fibroblasts from nine patients diagnosed with ECHS1 deficiency (ECHS1D)

IINTERVENTION

Loss of ECHS1 expression (knockout or pathogenic mutations)

OOUTCOME

Global gene expression (RNA-seq), proteomic changes, OXPHOS complex steady-state levels (western blot), OXPHOS enzyme activity, mitochondrial respiration, reactive oxygen species production, complex I assembly, and ECHS1 protein interactions (co-immunoprecipitation)

STUDY TYPE

other

SPECIALTY

mitochondrial medicine

SUMMARISED BY

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ORIGINAL PAPER

PMC10087869

Loss of mitochondrial fatty acid β‐oxidation protein short‐chain Enoyl‐CoA hydratase disrupts oxidative phosphorylation protein complex stability and function

The Febs Journal

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ABSTRACT

Abstract Short‐chain enoyl‐CoA hydratase 1 (ECHS1) is involved in the second step of mitochondrial fatty acid β‐oxidation (FAO), catalysing the hydration of short‐chain enoyl‐CoA esters to short‐chain 3‐hyroxyl‐CoA esters. Genetic deficiency in ECHS1 (ECHS1D) is associated with a specific subset of Leigh Syndrome, a disease typically caused by defects in oxidative phosphorylation (OXPHOS). Here, we examined the molecular pathogenesis of ECHS1D using a CRISPR/Cas9 edited human cell ‘knockout’ model and fibroblasts from ECHS1D patients. Transcriptome analysis of ECHS1 ‘knockout’ cells showed reductions in key mitochondrial pathways, including the tricarboxylic acid cycle, receptor‐mediated mitophagy and nucleotide biosynthesis. Subsequent proteomic analyses confirmed these reductions and revealed additional defects in mitochondrial oxidoreductase activity and fatty acid β‐oxidation. Functional analysis of ECHS1 ‘knockout’ cells showed reduced mitochondrial oxygen consumption rates when metabolising glucose or OXPHOS complex I‐linked substrates, as well as decreased complex I and complex IV enzyme activities. ECHS1 ‘knockout’ cells also exhibited decreased OXPHOS protein complex steady‐state levels (complex I, complex III 2 , complex IV, complex V and supercomplexes CIII 2 /CIV and CI/CIII 2 /CIV), which were associated with a defect in complex I assembly. Patient fibroblasts exhibit varied reduction of mature OXPHOS complex steady‐state levels, with defects detected in CIII 2 , CIV, CV and the CI/CIII 2 /CIV supercomplex. Overall, these findings highlight the contribution of defective OXPHOS function, in particular complex I deficiency, to the molecular pathogenesis of ECHS1D. Keywords: ECHS1 deficiency, fatty acid oxidation, mitochondria, mitochondrial disease, OXPHOS, short‐chain enoyl‐CoA hydratase

CITATION EXPORT

Harrison Burgin, Alice J Sharpe, Shuai Nie et al.. Loss of mitochondrial fatty acid β‐oxidation protein short‐chain Enoyl‐CoA hydratase disrupts oxidative phosphorylation protein complex stability and function. The Febs Journal. (2022). https://doi.org/10.1111/febs.16595

AUTHORS

10 · 10 CENTRES

Harrison Burgin, Alice J Sharpe, Shuai Nie, Mark Ziemann, Jordan J Crameri, Diana Stojanovski, James Pitt, Akira Ohtake, Kei Murayama, Matthew McKenzie

1 School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Geelong, Australia
2 Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia
3 Melbourne Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Australia
4 Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Australia
5 Department of Paediatrics, Victorian Clinical Genetics Services, Murdoch Childrens Research Institute, The University of Melbourne, Australia
6 Department of Pediatrics & Clinical Genomics, Faculty of Medicine, Saitama Medical University, Japan
7 Centre for Intractable Diseases, Saitama Medical University Hospital, Japan
8 Department of Metabolism, Chiba Children's Hospital, Japan
9 Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Melbourne, Australia
10 Department of Molecular and Translational Science, Monash University, Melbourne, Australia