TMP-Boc3Arg

Amino Acid Sequence

3D Structure (/structures/AF-P00374-F1-model_v6.cif)

Function

Catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate in a NADPH-dependent manner . Key enzyme in folate metabolism. Contributes to the nuclear and mitochondrial de novo thymidylate biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFR2.

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

B3A-induced degradation is blocked by proteasome inhibitors such as bortezomib and MG132 19. However, these inhibitors do not distinguish the core 20S proteasome from the 26S proteasome that is responsible for the degradation of ubiquitinated proteins. The 26S proteasome contains the 19S regulatory complex, which includes the DUBs USP14 and UCH37. Inhibition of these DUBs by the small molecule b-AP15 leads to inhibition of 26S proteasome 27. Therefore, we investigated the degradation of eDHFR in Cos1 cell lysates treated with b-AP15 to determine if B3A-induced degradation required the 26S proteasome. Inhibition of UCH37 was confirmed by activity profiling (Supplementary Figure S2b). TMP-B3A induced the degradation of eDHFR similarly in both untreated and b-AP15 treated lysates (Figure 2e). No accumulation of polyubiquitinated eDHFR was observed. These observations strongly suggest that B3A-mediated eDHFR degradation does not involve the ubiquitin-26S proteasome pathway.

Description

Therefore we investigated the degradation of eDHFR in Cos1 cell lysates treated with b-AP15 to determine if B3A-induced degradation required the 26S proteasome. Inhibition of UCH37 was confirmed by activity profiling (Supplementary Figure S2b). TMP-B3A induced the degradation of eDHFR similarly in both untreated and b-AP15 treated lysates (Figure 2e). E) b-AP15 does not block B3A-induced degradation of eDHFR. A Cos1 cell lysate (1 mg/ml) was preincubated with b-AP15 (20 μM) for 20 min before addition of eDHFR (800nM) and TMP-B3A (1.6 μM) were added to the assay.