Fig 2: Labile iron availability and oxidative stress control ISC synthesis and ACO1/IREBP1 protein function. (A) When intracellular labile iron is available, ISCs are synthesized by the cell and incorporated into apo-proteins, such as ACO1/IREBP1.18,23 Depletion of labile Fe2+ or an increase in cellular oxidative stress can cause disruption of ISC synthesis or oxidation of ACO1/IREBP's [4Fe-4S] cluster into an [3Fe-4S] cluster, inactivating the enzymatic function of ACO1.76 Figure created using Biorender.com. (B) Loss of an ISC cofactor from the bifunctional protein ACO1/IREBP1 precipitates the iron starvation response through iron responsive element binding activity, leading to an increase in cytosolic labile iron.18,23 This iron responsive element binding activity is also carried out by the protein IREBP2, however IREBP2 lacks both an ISC and aconitase activity.20,21 (1) Loss of a functional [4Fe-4S] leads to IREBP1 binding to IREs on mRNA. Binding (2) inhibits translation of FTH1 and (3) stabilizes the mRNA of TFR1.18,23 Figure created using Biorender.com. (C) The STRING database shows protein networks involved in ISC synthesis, ISC cellular trafficking, and iron regulation. The network was limited to the listed query proteins ACO1, ACO2, BOLA1, AIFM2 (also known as FSP1), BOLA2, BOLA3, CISD3, FDX1, FDX2, FXN, GLRX, GLRX3, GLRX5, GRPEL1, HSPA9, IREB2 (also known as IREBP2), ISCA1, ISCA2, ISCU, LYRM4 (also known as ISD11), MFN2, NFS1, NFU1, and PCBP1, and the confidence cutoff for interaction links was set to “medium” (0.400). The pink lines represent experimentally determined interactions between proteins involved in the synthesis, maturation, and cellular trafficking of iron-sulfur clusters. The purple line denotes homology between the proteins ISCA1 and ISCA2. Corresponding functions of the proteins included in the STRING networks include the following: ACO1 is a bifunctional protein, comprising an IREBP bound to an ISC cofactor that acts either as an enzyme or an iron regulatory protein.18,23 ACO2 is the mitochondrial form of aconitase which functions in the TCA cycle.77 IREBP2 (also known as IREB2) is homologous to IREBP1 and also functions in cellular iron regulation.21 AIFM2 (also known as FSP1) performs oxidoreductase activity to regenerate ubiquinol from ubiquinone and protects cellular lipids from oxidative radicals.78 ISCU serves as a protein scaffold on which ISCs are synthesized.17,37 During ISC synthesis, FXN facilitates incorporation of iron into the ISC and also promotes the transfer of sulfur atoms, reduced to sulfide anions, to the ISCU scaffold by NFS1.39,42 NFS1, which forms a complex with LYRM4, (also known as ISD11) during electron transfer, is known to be necessary for ISC synthesis.79 FDX1 and FDX2 function in ISC synthesis by reducing sulfur atoms, which are donated by NFS1, to sulfide anions.16,39 Knockdown of CISD3 leads to system Xc−/cysteine-induced iron accumulation and cell death via ferroptosis in tumor cells.80 The proteins ISCA1 and ISCA2 are vital to the maturation of ISCs from [2Fe-2S] clusters into [4Fe-4S] clusters that can be incorporated into apo-proteins.40 BOLA1 is a mitochondrial protein that interacts with GLRX5 and functions to prevent morphological changes to the mitochondria under oxidative stress.81 GLRX5 is a mitochondrial ISC chaperone protein that interacts with HSPA9 and GRPEL1 protein to perform Fe-S cluster transfer to ISC-dependent proteins.16,41,82 BOLA2 is a cytosolic chaperone for [2Fe-2S] clusters while complexed with GLRX3 or with PCBP1.38,83 PCBP1 is also capable of delivering cellular iron to ferritin for storage.84 BOLA3 is a chaperone protein that complexes with GLRX5 to perform ISC transport and incorporation into vital cellular proteins, including complexes of the mitochondrial electron transport chain.85,86 NFU1 functions in ISC synthesis by receiving [4Fe-4S] clusters from the BOLA3-GLRX5 complex and interacting with ISCA1 and ISCA2 during [4Fe-4S] maturation, and it may also have a role involving close interaction with the ISC scaffold protein ISCU.40 GLRX supplies electrons to ribonucleotide reductase and functions to reduce ROS in the cytosol.87 MFN2 is known to mediate mitochondrial fusion under conditions of mitochondrial and cellular stress.30,35 Figure created using string-db.org.

Fig 3: FECD endothelial tissues show differential expression of proteins necessary for ISC synthesis, mitochondrial fusion, and iron regulation compared to control donor corneal endothelial tissues. (A) ACO1/IREBP1 protein expression is 1.42-fold higher in FECD than in control lysates. (B) ACO2 expression is 1.42-fold lower in FECD than in control tissues; (C) FDX1 expression is 1.57-fold lower in FECD than in control tissues; (D) IREBP2 protein expression is 8.2-fold higher in FECD than in control lysates. (E) MFN2 expression is 1.2-fold lower in FECD than in control tissues; (F) NFS1 expression is twofold higher in FECD than in control tissues; and (G) PCBP1 shows a nonsignificant, 1.2-fold increase in expression in FECD compared to control tissues. Violet-blue dots represent control tissues and magenta dots represent FECD tissues. Error bars correspond to the mean and SEM; *P < 0.05, **P < 0.01, ***P < 0.001 regarding statistical significance, when compared to the control tissue pool. See Supplementary Figure S2 for bands.

Fig 4: FECD surgical tissues show decreased expression of genes responsible for ISC synthesis and trafficking. (A) RNA-seq analysis demonstrates downregulation of genes responsible for ISC synthesis and cellular ISC trafficking across a broad population. The location where the representative dataset was collected (Mayo, Russia, or UTSW) and mutation type (Control, no TCF4 repeats [Control] or FECD, with TCF4 repeats [FECD]) are shown for each sample. Gene set enrichment analysis showed downregulastion of an ISC gene signature in FECD patients compared to control patients. For FDR < 0.01, downregulation was noted in genes ACO2, BOLA1, FTH1, FDX1, GRPEL1, ISCA1, HSPA9, and PCBP1. For FDR < 0.05, downregulation was noted in the additional genes AIFM2, BOLA3, FXN, GLRX, GLRX5, and NFS1. (B) Quantitative PCR shows significantly decreased transcription of genes necessary for ISC synthesis. Violet-blue dots represent control tissues and magenta dots represent FECD tissues. Error bars correspond to the mean and SEM; *P < 0.05, **P < 0.01, ***P < 0.001 regarding statistical significance between the FECD group and the Control group. C) A subset of murine genes involved in ISC synthesis, Fdx1, Hspa9, Iscu, Nfs1, and Pcbp1, showed no significant difference in expression between homozygous Col8a2Q455K mouse models of early-stage FECD and homozygous Col8a2 wild-type controls.