ADC4EA

Amino Acid Sequence

3D Structure (/structures/AF-P09211-F1-model_v6.cif)

Function

Catalyzes conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. Involved in the formation of glutathione conjugates of both prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2). Participates in the formation of novel hepoxilin regioisomers. Acts as a negative regulator of ferroptosis by mediating glutathione conjugation and detoxification of 4-hydroxynonenal (4-HNE) reactive aldehyde. Negatively regulates CDK5 activity via p25/p35 translocation to prevent neurodegeneration.

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

when the cells were cotreated with bafilomycin A1, a specific inhibitor of vacuolar type H (+)-ATPase that prevents autophagy at late stage by inhibiting fusion between autophagosomes and lysosomes, the ADC4EA induced GSTP degradation was also greatly reversed. This suggested that in addition to UPP, ADC4EA can also induce GSTP protein degradation through autophagy pathway.

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

To further validate that this degradation was processed through the ubiquitin-proteasome pathway (UPP), we used bortezomib, a 20S proteasome inhibitor, to cotreat the cells, and as expected, the degradation induced by ADC4EA was significantly reversed. In addition, the degradation can also be reversed by PYR-41, which is an inhibitor of ubiquitin-activating enzyme E1, suggesting the degradation relies on the ubiquitination of the target protein (Figure 5).

Description

The most potent compound was ADC4EA, which generated over 80% clearance of GSTP protein at 40 μM.

Description

we further measured the apoptotic population in the treated cells using flow cytometry. Dose-dependently, ADC4EA induced apoptosis in cells after treatment for 3 h (Figure 7A) . Treatment with 80 μM of ADC4EA has generated 61% apoptotic popular.