Biology subjects

Labrecque, C. L.

Publications and source records attributed to Labrecque, C. L..

2 recordsLinked to original sources

bioRxiv2026

Revealing interactions between glutathione peroxidase 4 and phosphoinositides

Glutathione peroxidase 4 (GPx4) is the primary enzyme reducing lipid hydroperoxides, preventing membrane oxidative damage and protecting against ferroptosis. GPx4 is known to engage with lipid headgroups through electrostatic interactions, positioning the substrate for reduction. This work reveals and characterizes binding of highly anionic phosphoinositides (PIP lipids) by GPx4. PIPs are vital lipids in human cells and are central to many signaling processes, particularly in cytosolic facing membranes. Lipid overlay assays confirm interactions between GPx4 and phosphorylated PIPs, comparable to known anionic lipid binders. Protein NMR describes the interaction between GPx4 and PIPs within micelles. The greatest resonance shifting occurs with trisphosphorylated PIP, suggesting that higher anionic charge leads to greater binding, a known driver of GPx4 substrate recognition. Preferred anionic interactions were also confirmed with titration and crystallographic structure analysis of inositol phosphate 4 (IP4). A headgroup-binding site on GPx4 is revealed to be proximal to the cationic membrane interaction site. In conjunction with molecular simulations, these results show that PIP lipid interactions allow full engagement of GPx4 with the membrane and positions the headgroup to allow the lipid tail to interact with the catalytic site. Understanding whether GPx4 preferentially interacts with PIPs will allow better understanding of the protective function of this essential enzyme and a mechanism that may protect essential lipid signaling pathways from oxidative damage. SignificanceThis study allows a deeper structural and mechanistic understanding of GPx4, the primary enzyme that reduces lipid hydroperoxides and prevents ferroptosis. Gaining an understanding of phosphoinositide binding to GPx4 reveals a mechanism for potential preservation of these important signaling molecules and for ferroptosis protection. Observation of a specific binding site for headgroup engagement reveals a plausible lipid interaction mode and functional mechanism of this important cytoprotective enzyme.

biophysics↗

bioRxiv2024

Ligandability at the membrane interface of GPx4 revealed through a reverse micelle fragment screening platform

While they account for a large portion of drug targets, membrane proteins (MPs) present a unique challenge for drug discovery. Peripheral membrane proteins (PMPs), a class of proteins that bind reversibly to membranes, are also difficult targets, particularly those that function only while bound to membranes. The protein-membrane interface in PMPs is often where functional interactions and catalysis occur, making it a logical target for inhibition. However, interfaces are underexplored spaces in inhibitor design and there is a need for enhanced methods for small-molecule ligand discovery. In an effort to better initiate drug discovery efforts for PMPs, this study presents a screening methodology using membrane-mimicking reverse micelles (mmRM) and NMR-based fragment screening to assess ligandability in the protein-membrane interface. The proof-of-principle target, glutathione peroxidase 4 (GPx4), is a lipid hydroperoxidase which is essential for the oxidative protection of membranes and thereby the prevention of ferroptosis. GPx4 inhibition is promising for therapy-resistant cancer therapy, but current inhibitors are generally covalent ligands with limited clinical utility. Presented here is the discovery of non-covalent small-molecule ligands for membrane-bound GPx4 revealed through the mmRM fragment screening methodology. The fragments were tested against GPx4 in bulk aqueous conditions and displayed little to no binding to the protein without embedment into the membrane. The 9 hits had varying affinities and partitioning coefficients and revealed properties of fragments that bind within the protein-membrane interface. Additionally, a secondary screen confirmed the potential to progress the fragments by enhancing the affinity from > 200 {micro}M to [~]15 {micro}M with the addition of certain hydrophobic groups. This study presents an advancement of screening capabilities for membrane associated proteins, reveals ligandability within the GPx4 protein-membrane interface, and may serve as a starting point for developing non-covalent inhibitors of GPx4.

biophysics↗