CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes
Nunziata Maio, … , Tracey A. Rouault, Carsten G. Bönnemann
Nunziata Maio, … , Tracey A. Rouault, Carsten G. Bönnemann
Published June 17, 2024
Citation Information: J Clin Invest. 2024;134(12):e179559. https://doi.org/10.1172/JCI179559.
View: Text | PDF
Research Article Metabolism Muscle biology

CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes

Abstract

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

Authors

Nunziata Maio, Rotem Orbach, Irina T. Zaharieva, Ana Töpf, Sandra Donkervoort, Pinki Munot, Juliane Mueller, Tracey Willis, Sumit Verma, Stojan Peric, Deepa Krishnakumar, Sniya Sudhakar, A. Reghan Foley, Sarah Silverstein, Ganka Douglas, Lynn Pais, Stephanie DiTroia, Christopher Grunseich, Ying Hu, Caroline Sewry, Anna Sarkozy, Volker Straub, Francesco Muntoni, Tracey A. Rouault, Carsten G. Bönnemann

×

Figure 4

The CIAO1 variants identified in P1 cause protein instability and compromised biogenesis of multiple Fe-S clients of the CIA complex.

Options: View larger image (or click on image) Download as PowerPoint
The CIAO1 variants identified in P1 cause protein instability and compro...
(A and B) Levels of the CIA components and Fe-S proteins in P1- and parent-derived fibroblasts (“Fath” and “Moth” correspond to father and mother of P1, respectively). Levels of FAM96A are also shown, along with the cytosolic iron and ISC chaperone BOLA2 (46). α-Tubulin (α-TUB) was included as a loading control and is presented again in panel G. To avoid reprobing of the same blotting membrane, the same lysates were run on adjacent wells on the gel shown in Supplemental Figure 4A, and α-tubulin was probed only once for the set of samples. (C) Top left corner shows the reaction catalyzed by DPYD. Top right corner is a ribbon representation of the crystal structure of DPYD (Protein Data Bank [PDB] ID: 1H7W), which assembles into a dimer containing a total of 8 [4Fe-4S] clusters. Bottom section shows DPYD-mediated conversion of [4-14C]-thymine to [4-14C]-dihydrothymine in lysates derived from P1 or control cells assayed by TLC and autoradiography. The reaction mix containing [4-14C]-T alone (no extract) was loaded to visualize the substrate (4-14C-thymine). (D) 55Fe incorporation into POLD1-FLAG/MYC expressed for 16 hours in P1 and parental fibroblasts. Anti-FLAG IB shows equal amounts of POLD1-F/M immunoprecipitated (A–D, n = 4 biological replicates). (E) Quantification by scintillation counter of 55Fe incorporated into POLD1-F/M. [55Fe]-POLD1-F/M levels in control cells (father of P1) were quantified and set to 100%. Values are expressed as a percentage of control and are given as the mean ± SEM. ****P < 0.0001, by 1-way ANOVA Šidák’s multiple-comparison test for P1 versus the father and P1 versus the mother. n = 4 biological replicates. (F) In-gel activity assays of cytosolic (ACO1) and mitochondrial (ACO2) aconitases in fibroblasts from P1 compared with control cells. (G) IBs to detect IRP1 and IRP2, TFRC, FTH, FTL, GLRX3, and ALAD on lysates from P1- and parent-derived fibroblasts. (H) Levels of FBXL5 in P1 and parental cells (F and G, n = 3 biological replicates). (I) Iron content in P1- and parent-derived mitochondria as assessed by ICP-MS (n = 3 biological replicates). No statistically significant difference was detected between experimental groups by 1-way ANOVA Šidák’s multiple-comparison test.