N15

Amino Acid Sequence

3D Structure (/structures/struct-20261023-171611-4decd5.cif)

Function

Essential antioxidant peroxidase that directly reduces phospholipid hydroperoxide even if they are incorporated in membranes and lipoproteins. Can also reduce cholesterol hydroperoxide and thymine hydroperoxide (By similarity). Plays a key role in protecting cells from oxidative damage by preventing membrane lipid peroxidation. Required to prevent cells from ferroptosis, a non-apoptotic cell death resulting from an iron-dependent accumulation of lipid reactive oxygen species. The presence of selenocysteine (Sec) versus Cys at the active site is essential for life: it provides resistance to overoxidation and prevents cells against ferroptosis (By similarity). The presence of Sec at the active site is also essential for the survival of a specific type of parvalbumin-positive interneurons, thereby preventing against fatal epileptic seizures (By similarity). May be required to protect cells from the toxicity of ingested lipid hydroperoxides (By similarity). Required for normal sperm development and male fertility (By similarity). Essential for maturation and survival of photoreceptor cells (By similarity). Plays a role in a primary T-cell response to viral and parasitic infection by protecting T-cells from ferroptosis and by supporting T-cell expansion (By similarity). Plays a role of glutathione peroxidase in platelets in the arachidonic acid metabolism. Reduces hydroperoxy ester lipids formed by a 15-lipoxygenase that may play a role as down-regulator of the cellular 15-lipoxygenase pathway (By similarity). Can reduce fatty acid-derived hydroperoxides. Can also reduce small soluble hydroperoxides such as H2O2, cumene hydroperoxide and tert-butyl hydroperoxide.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

Encouraged by its strong degradation potency, we conducted a series of experiments to elucidate the molecular mechanism underlying N15- mediated GPX4 degradation. By the co-incubation of a well-recognized protein synthesis inhibitor, cycloheximide (CHX), we observed that N15-treatment dramatically accelerated the depletion of existing GPX4 protein pools, reducing its degradation half-life from over 24 h to approximately 8 h (Fig. 5A–B). Taking into account the role of CHX impeding protein translation, N15 may promote the degradation of pre- existing GPX4 via the ubiquitin-proteasome system (UPS) pathway in a time-dependent manner. To gain deeper understanding of the degradation mechanism of N15, we investigated the effect of well-employed inhibitors targeting different cellular protein clearance systems. As shown in Fig. 5C–D, the pre-treatment with proteasome inhibitor MG132 completely blocked the GPX4 degradation induced by N15, whereas the lysosomal inhibitor chloroquine (CQ) showed trace protective effect. The entire abolition of degradation by MG132 treatment demonstrates that the ubiquitin- proteasome system is primarily responsible for the performance of N15. To further substantiate this mechanism, we performed immunoprecipitation (IP) experiments in HT1080 cells. After treatment with N15 (100 nM, 2.5 h), GPX4 was immunoprecipitated, and ubiquitination was assessed by immunoblotting. As shown in Fig. S7, N15 markedly enhanced the level of cellular ubiquitination, thereby providing direct evidence that GPX4 degradation occurs via the UPS pathway. These comparative results provide definitive evidences that N15 functions as a degrader by specifically recruiting GPX4 to the ubiquitin-proteasome pathway for subsequent destruction.

Description

Given the potent anticancer activity and degradation efficacy of N15, we further investigated its effect on GPX4 degradation at various concentrations and time points, which presented both concentration- and time-dependent degradation profiles. Quantitative analysis revealed that N15 mediates GPX4 depletion with a DC50 of 0.028 μM following 10 h of treatment (Fig. 4A and B).

Description

we evaluated the antiproliferative activities of all synthesized GPX4 HyT degraders against ferroptosis-sensitive human fibrosarcoma HT1080 cells using the standard MTT assay wherein ML162 served as the reference compound for comparison. For amide-linking compounds N10–N23, similar trend was found to the PEG series, alkyl chain length significantly influenced their antiproliferative activity. Compound N15, featuring a 10-methylene linker, demonstrated optimal cytotoxicity (IC50 = 3.1 nM);