EA-Boc3Arg

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. 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.

Amino Acid Sequence

3D Structure (/structures/AF-P08263-F1-model_v6.cif)

Function

Glutathione S-transferase that catalyzes the nucleophilic attack of the sulfur atom of glutathione on the electrophilic groups of a wide range of exogenous and endogenous compounds (Probable). Involved in the formation of glutathione conjugates of both prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2). It also catalyzes the isomerization of D5-androstene-3,17-dione (AD) into D4-androstene-3,17-dione and may therefore play an important role in hormone biosynthesis. Through its glutathione-dependent peroxidase activity toward the fatty acid hydroperoxide (13S)-hydroperoxy-(9Z,11E)-octadecadienoate/13-HPODE it is also involved in the metabolism of oxidized linoleic acid.

Mechanism

The ubiquitin-independent proteasome pathway (UIPP) refers to a pathway in which proteins are directly degraded by the 20S proteasome without reliance on ubiquitin tagging. Boc3Arg-containing HyT can link to target proteins through a target protein-recognizing ligand; Boc3Arg directly binds to and activates the 20S proteasome, forming a ternary complex. Target proteins are directly degraded by the 20S proteasome, and this process is independent of ubiquitination, ATP, and the 26S proteasome.

Description

Therefore, we investigated whether Boc3Arg-induced degradation also utilized the ubiquitin-proteasome system. The specific proteasome inhibitor bortezomib blocks the Boc3Arg-mediated degradation of eDHFR and endogenous GST-α1-GFP in whole cells (Figures 4A and 5A). Lactacystin, a second specific proteasome inhibitor, blocks Boc3Arg-induced degradation in Cos-1 cell lysates (Figure 6A), as does the less specific inhibitor MG132 (Figure 6D). However, we failed to observe the accumulation of higher molecular weight forms of any of the proteins in the presence of proteasome inhibitors, even when deubiquitylating enzyme inhibitors were included with proteasome inhibitors. ATP is required both for ubiquitylation and for the degradation of ubiquitylated proteins by the 26S proteasome, but omission of the ATP regenerating system did not inhibit Boc3Arg-induced degradation (Figure 6B). Although it can often be difficult to detect ubiquitylation, these observations suggest that the Boc3Arg group induces degradation of the target protein by a mechanism that is independent of the ubiquitin pathways. (B) Degradation of GST-a1 (0.2 mg/ml) was modified by EA-Fur-Boc3Arg (40 mM) then diluted 50-fold into NIH 3T3 cell lysates. Closed diamonds, standard assay buffer containing ATP regenerating system;closed squares, ATP regenerating system omitted. (D) Effect of MG132 (100 mM) on the EA-Boc3Arg-induced degradation of GST-a1 in Cos-1 cell lysates supple mented with an ATP regeneration system

Description

Treatment with EA-Boc3Arg caused the rapid and robust degradation of eDHFR-HA-GST-α1. Degradation was dose-dependent, with 10 μM causing ∼50% degradation in 1.2 hr (Figure 5D).

Description

The eDHFR-HA-GST-α1 fusion protein allows direct comparison of degradation induced by EA-Boc3Arg and TMP-Boc3Arg. While 80 μM EA-Boc3Arg induces nearly complete degradation of the fusion protein in under 2 h, only ∼25% degradation is observed in 5 h when cells are treated with the same concentration of TMP-Boc3Arg.