APL-PLA

Amino Acid Sequence

3D Structure (/structures/AF-P10275-F1-model_v6.cif)

Function

teroid hormone receptors are ligand-activated transcription factors that regulate eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Transcription factor activity is modulated by bound coactivator and corepressor proteins like ZBTB7A that recruits NCOR1 and NCOR2 to the androgen response elements/ARE on target genes, negatively regulating androgen receptor signaling and androgen-induced cell proliferation. Transcription activation is also down-regulated by NR0B2. Activated, but not phosphorylated, by HIPK3 and ZIPK/DAPK3.

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 evaluated the accumulation of polyubiquitin chains in cells, both with and without pretreatment with epoxomicin. It has been reported that epoxomicin treatment inhibits protein degradation, leading to the accumulation of polyubiquitin chains.32 Consistent with our previous experiment, during normal proteolysis with ARL-PLA and ARL-VHL, no accumulation of polyubiquitin chains was observed due to their complete degradation. However, upon treatment with epoxomicin, a statistically significant accumulation of polyubiquitin chains was observed in cells treated with ARL-PLA and ARL-VHL (Figure 2d,e) To confirm the polyubiquitination of AR, we performed an immunoprecipitation (IP) assay to identify polyubiquitinated AR. By conducting IP with an AR antibody, we successfully precipitated polyubiquitinated AR. The polyubiquitin chains and AR were subsequently analyzed by Western blot using FK2 as the antibody for polyubiquitin chains and an AR antibody. Our findings revealed that polyubiquitin chains increased in the cells upon treatment with epoxomicin, a proteasome inhibitor, even after treatment with ARL-PLA and ARL-VHL (Figure 2f). This result suggests that ARL-PLA treatment induces the polyubiquitination of AR, leading to its proteasome-mediated degradation.

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

previous research has indicated that PLA can induce autophagosome formation by increasing LC3B II levels.26 Therefore, we explored whether ARL-PLA could also induce targeted protein degradation through an alternative pathway, specifically the autophagy-lysosomal pathway. We first investigated AR degradation by treating cells with wortmannin, an inhibitor of autophagosome formation. In the absence of wortmannin, both ARL-adm and ARL-PLA effectively induced AR degradation. However, when wortmannin was present, AR degradation was significantly reduced. These results suggest that ARL-PLA degrades AR via both the autophagy and proteasome pathways. Furthermore, we confirmed that ARL-PLA induces the formation of autophagosomes, as evidenced by an increase in the intracellular accumulation of autophagic vacuoles, monitored using the autophagosome marker Monodansylcadaverine (MDC).33 Upon ARL-PLA treatment, the fluorescence intensity of MDC-labeled autophagosomes increased compared to controls, indicating that AR is specifically targeted and degraded through the autophagy pathway. Following autophagosome formation, the proteins are degraded through fusion with lysosomes.34 To further explore this, we treated cells with Bafilomycin A1, an inhibitor of lysosome fusion, which leads to the accumulation of autophagosomes in the cell.35 Treatment with Bafilomycin A1 increased the turnover rate of LC3B II, which forms the autophagy membrane, in ARL-PLA-treated cells. These findings suggest that AR, captured by ARL-PLA, is ultimately degraded through the autophagy-lysosomal pathway.

Description

When the concentration of ARL-PLA was increased, a more significant amount of AR degradation was observed. From this concentration-dependent AR degradation, we calculated a DC50 of 18.25 μM