FDX1 regulates cellular protein lipoylation through direct binding to LIAS

Abstract

Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 regulates protein lipoylation by directly binding to the lipoyl synthase (LIAS) enzyme and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss-of-function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling of cells growing in either normal or low glucose conditions established that FDX1 loss-of-function results in the induction of both compensatory metabolism related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-functions is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.

Fig. 1. Genetic evidence supporting the role of FDX1 in regulating protein lipoylation.

(A) FDX1 mRNA expression levels in cancer cell lines from the CCLE categorized by lineage. (B) The CRISPR/Cas9 FDX1 loss-of-function viability outcome in 1128 cell lines correlated to the loss-of-function of all other tested genes. Plotted are the correlation values and the p-value for the 1000 most significant correlations. In green are genes associated with the protein lipoylation pathway. (C) The correlation of LIAS and FDX1 CRISPR/Cas9 loss-of-function across 1128 cell lines. (D) Top molecular function (GO) enriched categories in the 1000 genes that have their loss-of-function most correlated with FDX1 loss-of-function. (E) Indicated proteins were analyzed using immunoblot assays of HEK293T, K562 and ABC1 WT cells or cells with CRISPR/Cas9 induced FDX1 loss-of-function (KO) alone or with reconstituted FDX1 (KO+OE). The data used to generate plots A-D is provided as supplementary table 1.

Fig. 2. FDX1 loss-of-function induces the integrated stress response and inhibits proliferation and respiration in low glucose conditions.

(A) schematic of the experimental conditions. (B) The oxygen consumption rate (OCR) was detected in ABC1 WT, FDX1 KO and FDX1 KO cells with FDX1 expression reconstituted and after the addition of oligomycin (ATP-linked), the uncoupler FCCP (maximal), or the electron transport inhibitor antimycin A/rotenone (baseline) (mean ± SD, of at least 4 biological replicates). (C) Basal OCR was measured in HEK293T WT, FDX1 KO and FDX1 KO cells with FDX1 expression reconstituted (mean ± SD, of at least 8 biological replicates). (D) Cell proliferation as measured by confluency is shown for HEK293T WT and FDX1 KO cells grown in either 10mM or 2mM glucose containing media. (n=3) (E-F) PCA of gene expression levels derived from RNA-seq in ABC1 (E) and K562 (F) WT (gray) and FDX1 KO (green) cells growing in either 2mM (square) or 10mM (circle) glucose conditions (n=2 for each condition). (G-J) Volcano plots relating to LOG2 fold-change (x-axis) and - LOG10(p-values) (y-axis) for gene expression changes observed in FDX1 KO as compared to WT. Volcano plots for K562 (G, H) and ABC1 (I, J) show cells growing in media containing either 10mM (G, I) or 2mM (H,J) glucose. (K-N) Gene set enrichment analysis (GSEA) of GO_Biological Processes using either PC1 (K, M) or PC2 (L, N) weights as the gene ranks. Representative categories were selected, and full data is provided in Table S2.

Fig. 3. FDX1 loss-of-function results in metabolite signature consistent with protein lipoylation deficiency.

(A) Schematic of the metabolic processes where lipoylated proteins are involved. (B) LOG2 fold change of metabolites in the media of either ABC1, HEK293T or K562 WT as compared to FDX1 loss-of function cells grown in media containing either 2mM (y-axis) or 10mM (x-axis) glucose. (C-D) changes in media pyruvate (C) or serine (D) levels in either WT, FDX1 loss-of-function (KO), or FDX1 loss-of-function cells with reconstituted FDX1 (KO+OE) HEK293T, ABC1 and K562 cells. (E-G) Intracellular metabolite level changes between WT and FDX1 loss-of-function cells (KO) ABC1 (E), HEK293T (F) and K562 (G) cells grown in media containing either 2mM (y-axis) or 10mM (x-axis) glucose. (H-L) Changes in intracellular levels of C5- (H) or C3- (I) carnitines or phospho-ethanolamine (K) or CDP-choline (L) in either WT, FDX1 loss-of-function (KO), or FDX1 loss-of-function cells with reconstituted FDX1 (KO+OE) HEK293T, ABC1 and K562 cells. Schematic of relevant pathways is presented (J).

Fig. 4. FDX1 directly binds LIAS in cells and in vitro.

(A) Schematic describing the nano-luciferase complementation assays used and the equation used to normalize luminescence ratio (NLR) score corresponding to the raw luminescence value of the tested pair (A–B) divided by the sum of luminescence value from the two individual proteins (A-alone + B-alone). (B) The oligomerization of FDX1 was measured by overexpressing FDX1 tagged with fragment 1 of the complementation Nano-luciferase (C1) together with either FDX1 tagged with fragment 2 (C2) or FDX2 tagged with fragment 2 (C2) used as a negative control. Luminescence was measured 24 h after transfection (n = 3) (C) FDX1 or FDX2 used as a control tagged with fragment 1 of the complementation Nano-luciferase (C1) were overexpressed in the presence of indicated proteins tagged with fragment 2 of the complementation Nano-luciferase (C2). Luminescence was measured 24 h after transfection (n = 3). (B-C) Calculated NLR score is presented. (D) HEK293T cell lysates were subjected to a 5–30% glycerol gradient and FDX1, LIAS and GCSH protein sedimentation was analyzed using immunoblot analysis. (E) FDX1-C2 was overexpressed in HEK293T cells together with either FDX1-C1 or LIAS-C1 and lysates were analyzed for luminescence following sedimentation in 5–30% glycerol gradient. (F) MST results from titration of FDX1 to Red-NHS dye-labeled LIAS are shown, averaged raw data (n=3, black dots), the calculated binding curve fit from which the Kd was extracted (solid blue line), and the standard deviations (vertical black error bars). The fraction bound of labeled LIAS is plotted against the concentration of titrated FDX1 ligand and a K_d of 6 ± 2 µM was determined.