Kwon, J. J. | Search bioRxiv

bioRxiv2025

Deep Mutational Scanning of FDX1 Identifies Key Structural Determinants of Lipoylation and Cuproptosis

Cuproptosis is a recently described form of regulated cell death triggered by ionophore-induced copper (Cu) overload in mitochondria. It is critically dependent on ferredoxin 1 (FDX1), a mitochondrial reductase that facilitates cuproptosis by reducing ionophore-bound Cu(II) to Cu(I) thereby triggering its release, and by promoting mitochondrial protein lipoylation, which is directly targeted by the released Cu to drive cell death. Despite the pivotal role of FDX1 in cuproptosis, the structural determinants underlying its distinct functions remain unclear. To address this, we performed deep mutational scanning (DMS) of FDX1 and identified two conserved, solvent-exposed residues--D136 and D139--on its third alpha helix (-helix 3) that are critical for both FDX1-mediated cuproptosis and lipoylation. Charge-reversal mutations at these positions abolished FDX1s ability to induce cuproptosis and support lipoylation in cells, despite retaining full enzymatic activity in vitro. Guided by structural and genomic analyses, we further identified dihydrolipoamide dehydrogenase (DLD), the E3 subunit of lipoylated complexes as an alternative FDX1 reductase both in cells and in vitro. Together, these findings establish the acidic -helix 3 of FDX1 as a critical interface for its upstream regulation and suggest that FDX1s roles in cuproptosis and in lipoylation are both structurally and functionally linked.

cell biology↗

bioRxiv2022

Engineered Serum Markers for Noninvasive Monitoring of Gene Expression in the Brain

Noninvasive efforts to map brain gene expression have been hampered by low sensitivity and limited access to the brain. Here, we introduce a new platform that enables multiplexed, noninvasive, and site-specific monitoring of brain gene expression through a novel class of engineered reporters called Released Markers of Activity (RMAs). Instead of detecting gene expression in the less accessible brain, RMA reporters exit from a known brain region into the blood, where they can be easily measured with biochemical techniques. Expressing RMAs at a single brain site, typically covering [~]1% of the brain volume, provides up to a 39,000-fold signal increase over the baseline in vivo. Further, expression of RMAs in as few as several hundred neurons was sufficient for their reliable detection. When placed under a promoter upregulated by neuronal activity, RMAs could be used to measure neuronal activity in specific brain regions with a simple blood draw. We found that chemogenetic activation of cells expressing Fos-responsive RMA increased serum levels of RMA over 4-fold compared to non-activated controls. By contrast, a control RMA expressed under a constitutive neuronal promoter did not show such upregulation, demonstrating multiplexed ratiometric measurement with RMAs and proving specificity of neuronal activity discrimination. Together, our study pioneers a new noninvasive paradigm for repeatable and multiplexed monitoring of gene expression in an intact brain with sensitivity that is currently unavailable through other noninvasive gene expression reporter systems.

neuroscience↗