Fig. 5: DLD can serve as an alternative upstream regulator of FDX1.

A Volcano plot shows the CRISPR/Cas9 FDX1 loss-of-function viability outcome in 1373 cell lines correlated with the loss of function of all other tested genes. Plotted are the correlation values and the p-value for the 17,915 genes (see Methods for details). Possible FDX1 upstream regulators are colored orange. B Schematic of an alternative upstream regulation of FDX1. C Immunoprecipitation (IP) with anti-Strep-Tag II (STII) of C-terminally STII-tagged DLD or FDXR stably expressed in HEK293T cells. Indicated FDX1 constructs are transiently transfected. pMT025 is the same expression vector used for the DMS screen, and “stop” indicates a stop codon (i.e., no tag). ORF, open reading frame. Endo, endogenous. D In vitro FDX1- and LIAS-mediated lipoylation activity assay that compare FDXR and DLD as the upstream reducing source and FDXR alone as control. Data is presented as mean ± SD of three replicates. E−G CRISPR/Cas12a mediated DLD KO in JHH7 (E) and in HEK293T (F), and FDXR KO in JHH7 (G). The lysates were immunoblotted with the indicated antibodies. Green arrows highlight changes in levels of key proteins of interest. H Top, HEK293T overexpressing the indicated constructs were assayed for viability after 2 days of ES-Cu treatment. Bottom, immunoblots of cell lines shown above. Viability data is presented as mean ± SEM of five biological replicates. I Schematic depicting our model: during homeostasis, FDX1 uses its negative charges on its third alpha helix to facilitate critical downstream steps including lipoylation and Cu reduction in ES (left panel). By flipping the charges of those residues (right panel), an inhibitory protein binds mutant FDX1, preventing it from receiving electrons from DLD, FDXR, and/or other reducing sources. This results in a functionally inactive FDX1.