7b

Amino Acid Sequence

3D Structure (/structures/struct-20261026-144328-b09795.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. May be required to protect cells from the toxicity of ingested lipid hydroperoxides. Required for normal sperm development and male fertility. Essential for maturation and survival of photoreceptor cells. 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. 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. Can reduce fatty acid-derived hydroperoxides. Can also reduce small soluble hydroperoxides such as H2O2, cumene hydroperoxide and tert-butyl hydroperoxide.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

We then investigated the mechanism of 7b-induced GPX4 degradation in HT1080 cells. We treated HT1080 cells with cycloheximide (CHX), a protein synthesis inhibitor combined with 7b [30]. Notably, 7b exhibited accelerated degradation kinetics when protein translation was inhibited, suggesting that 7b effectively induces the time-dependent degradation of GPX4 through the UPS (Fig. 4A,B). To gain deeper insights into the mechanism of 7b-mediated degradation of GPX4, we performed co-treatment experiments with MG132, a proteasome inhibitor, in combination with 7b. Our data demonstrated that MG132 only partially blocked the degradation of GPX4 (Fig. 4C,D). Previous reports showed that GPX4 degraders operate not only through the ubiquitin proteasome-mediated proteolysis pathway but also via the autophagy-lysosome pathway [23]. As shown in Fig. 4C, when MG132 and chloroquine (CQ) are administered in combination. The degradation of GPX4 induced by 7b was prevented, underscoring the involvement of both UPS and autophagy mechanisms in the degradation process.

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

We then investigated the mechanism of 7b-induced GPX4 degradation in HT1080 cells. We treated HT1080 cells with cycloheximide (CHX), a protein synthesis inhibitor combined with 7b [30]. Notably, 7b exhibited accelerated degradation kinetics when protein translation was inhibited, suggesting that 7b effectively induces the time-dependent degradation of GPX4 through the UPS (Fig. 4A,B). To gain deeper insights into the mechanism of 7b-mediated degradation of GPX4, we performed co-treatment experiments with MG132, a proteasome inhibitor, in combination with 7b. Our data demonstrated that MG132 only partially blocked the degradation of GPX4 (Fig. 4C,D). Previous reports showed that GPX4 degraders operate not only through the ubiquitin proteasome-mediated proteolysis pathway but also via the autophagy-lysosome pathway [23]. As shown in Fig. 4C, when MG132 and chloroquine (CQ) are administered in combination. The degradation of GPX4 induced by 7b was prevented, underscoring the involvement of both UPS and autophagy mechanisms in the degradation process.

Description

To further examine the effect of the linking group, we installed a 2-polyethylene glycol (2-PEG) linker conjugated to the adamantyl group, resulting in compound 7b. Compound 7b has an IC50 value of 0.13 μM, demonstrating slightly improved potency compared to that of ML210.

Description

The ability of 7b to induce the degradation of GPX4 protein in HT1080 cells was investigated by western blot assay (Fig. 3). The result suggested that 7b potently reduces GPX4 protein level in a dose-dependent manner, achieving a DC50 value of 0.057 μM, which was approximately 12-fold more potent than ML210.