YL2-HyT6

Amino Acid Sequence

3D Structure (/structures/AF-Q15788-F1-model_v6.cif)

Function

Nuclear receptor coactivator that directly binds nuclear receptors and stimulates the transcriptional activities in a hormone-dependent fashion. Involved in the coactivation of different nuclear receptors, such as for steroids (PGR, GR and ER), retinoids (RXRs), thyroid hormone (TRs) and prostanoids (PPARs). Also involved in coactivation mediated by STAT3, STAT5A, STAT5B and STAT6 transcription factors. Displays histone acetyltransferase activity toward H3 and H4; the relevance of such activity remains however unclear. Plays a central role in creating multisubunit coactivator complexes that act via remodeling of chromatin, and possibly acts by participating in both chromatin remodeling and recruitment of general transcription factors. Required with NCOA2 to control energy balance between white and brown adipose tissues. Required for mediating steroid hormone response. Isoform 2 has a higher thyroid hormone-dependent transactivation activity than isoform 1 and isoform 3.

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 test whether YL2-HyT6 induced SRC-1 degradation by the proteasome-dependent pathway, MDA-MB-231 cells were cotreated with YL2-HyT6 and MG-132, a proteasome inhibitor. As expected, SRC-1 levels were not changed in the presence of MG-132, suggesting that YL2-HyT6 degrades SRC through the proteasome-mediated process (Figure 6c).Protein degradation achieved by hijacking the unfolded protein pathway is known to be associated with molecular chaperones, such as heat shock proteins (HSPs). For example, Hsp70 recognizes exposed hydrophobic regions of unfolded or misfolded proteins and mediates polyubiquitination and proteasomal degradation. To examine this, cells were incubated with 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a well-known Hsp90 inhibitor that induces the upregulation of Hsp70 expression [39]. Immunoblot analysis showed that elevated levels of Hsp70 efficiently increased the cellular activity of YL2-HyT6, finally leading to SRC-1 degradation even at a concentration that did not affect SRC-1 levels previously (Figure 6d). These results were consistent with a previous study showing enhanced degradation activity toward the androgen receptor after cotreatment with an Hsp90 inhibitor [33]. These results indicate that SRC-1 degradation via hydrophobic tagging strategy can be induced through a chaperone-mediated ubiquitin proteasome system.

Description

Next, we conducted a competitive fluorescence polarization (FP) assay to measure the binding affinities of the synthesized compounds to SRC-1 (Figure 3b). Most compounds retained their abilities to bind to the recombinant PAS B domain of SRC-1. This result was in good agreement with CD results. Among them, YL2 HyT6 with a tri(ethylene glycol) linker exhibited the best binding affinity (Ki = 237 nM), which was compatible with that of the original stapled peptide YL2 (Ki = 137 nM).

Description

These results confirmed that the chimeric structure of YL2-HyT6 was essential for the hydrophobic-tagged strategy for targeted protein degradation. Notably, YL2-HyT6 reduced cellular SRC-1 levels with a DC50 value of ~5 µM (Figure S4), which is more than twice as good as that of ND1-YL2 (DC50 ≈ 10 µM)