Spearheaded by
@jeffreychsiao.bsky.social and Doug Warui 🙌 . Huge thanks to Jason Kwon for the computational analysis , Tamra, @maggiedreishpoon.bsky.social , Nolan Bick and
Squire Booker for making this possible.
@bidmc-cancercenter.bsky.social @broadinstitute.org

Key take-home points:
1.The acidic α-helix 3 is a crucial regulatory interface on FDX1
2. The roles of FDX1 in cuproptosis and lipoylation couldn't be uncoupled, suggesting they are a single, structurally linked function
3.DLD is a newly discovered upstream reductase for FDX1

Indeed, DLD bound FDX1 more strongly than FDXR in cells, and this interaction was lost with the D136R/D139R mutants. DLD could also replace FDXR in an FDX1-dependent lipoylation assay in vitro .

This hinted that FDX1 might need or have another electron donor in cells (which is not FDXR). Based on structural homology, we tested if DLD (dihydrolipoamide dehydrogenase), the E3 subunit of lipoylated complexes, could also serve as an FDX1 electron donor.

But there was a twist. Cells with D136R or D139R mutants were also completely deficient in lipoylation. Yet in vitro these mutants stayed fully functional — supporting LIAS-mediated lipoylation and also binding FDXR/LIAS with similar affinities as WT.

This analysis highlighted two residues — D136 and D139 on FDX1’s α-helix 3.

Mutating them to alanine left cuproptosis intact, but flipping their charge (Asp → Arg) made cells resistant to elesclomol–Cu–induced death.

This let us filter out mutations predicted to disrupt overall folding. We focused on variants that blocked FDX1-driven cuproptosis while predicted to remain structurally intact.

We paired the DMS data with an in silico FoldX analysis, which predicts the mean free-energy change (ΔΔG) for each residue in FDX1.

To find out, we performed deep mutational scanning (DMS), mutating every amino acid in FDX1 and testing its ability to promote cuproptosis in two cell line models.

So FDX1 has two roles: 1. Directly reducing Cu(II) bound to elesclomol, freeing Cu(I) and 2. Promoting protein lipoylation, the copper target in cuproptosis.
We set out to explore if these functions could be structurally uncoupled.

Later, we found FDX1 is also the key upstream regulator of protein #lipoylation, the very target of copper in cuproptosis. www.jbc.org/article/S002...

We previously showed that #FDX1 is essential for #cuproptosis , a copper-dependent cell death pathway where Cu targets lipoylated enzymes & Fe–S cluster proteins. www.science.org/doi/10.1126/...

Just had exactly the same experience - went to watch wicked with my girls -with the exact opposite outcome, so boring. lucky they had beer in the theater 😂 .