23

Amino Acid Sequence

3D Structure (/structures/AF-O60885-F1-model_v6.cif)

Function

Chromatin reader protein that recognizes and binds acetylated histones and plays a key role in transmission of epigenetic memory across cell divisions and transcription regulation. Remains associated with acetylated chromatin throughout the entire cell cycle and provides epigenetic memory for postmitotic G1 gene transcription by preserving acetylated chromatin status and maintaining high-order chromatin structure. During interphase, plays a key role in regulating the transcription of signal-inducible genes by associating with the P-TEFb complex and recruiting it to promoters. Also recruits P-TEFb complex to distal enhancers, so called anti-pause enhancers in collaboration with JMJD6. BRD4 and JMJD6 are required to form the transcriptionally active P-TEFb complex by displacing negative regulators such as HEXIM1 and 7SKsnRNA complex from P-TEFb, thereby transforming it into an active form that can then phosphorylate the C-terminal domain (CTD) of RNA polymerase II. Regulates differentiation of naive CD4+ T-cells into T-helper Th17 by promoting recruitment of P-TEFb to promoters (By similarity). Promotes phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II. According to a report, directly acts as an atypical protein kinase and mediates phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II; these data however need additional evidences in vivo. In addition to acetylated histones, also recognizes and binds acetylated RELA, leading to further recruitment of the P-TEFb complex and subsequent activation of NF-kappa-B. Also acts as a regulator of p53/TP53-mediated transcription: following phosphorylation by CK2, recruited to p53/TP53 specific target promoters.

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

To further characterize the mechanism of action of these two compounds, we investigated whether BRD4 degradation was based on proteasome or lysosome activity. Therefore, BRD4 levels were assessed in 4T1 cells preincubated (30 min) with pharmacologically active concentrations of the proteasome inhibitor bortezomib, or the lysosome inhibitor chloroquine, just before being exposed to compound26or compound23. As shown in Figure 6A, neither inhibitor affected the26-induced degradation of BRD4. In contrast, only chloroquine counter acted the effects of23on BRD4 protein levels, suggesting that this specific HyTag acts via the lysosomal pathway of degradation.

Mechanism

The unfolded protein response (UPR) is a quality-control mechanism triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. Under ER stress, the chaperone BiP/GRP78 dissociates from three ER transmembrane sensors—IRE1α, PERK, and ATF6—activating three distinct UPR branches. Activated IRE1α splices XBP1 mRNA to generate XBP1s. PERK phosphorylates eIF2α, attenuating global translation while selectively inducing ATF4. ATF6 translocates to the Golgi apparatus and is cleaved to release its active nuclear form, ATF6(N). These transcription factors coordinately upregulate chaperones, folding enzymes, ER-associated degradation (ERAD), and autophagy to restore cellular proteostasis. However, if the stress persists or remains unresolved, prolonged UPR signaling triggers apoptosis through the CHOP and BAX/BAK-caspase pathways.

Description

Three ER membrane-associated sensors are responsible for the activation of the UPR signaling pathway: activating transcription factor-6 (ATF6), double-stranded RNA-dependent protein kinase (PKR)-like eukaryotic initiation factor 2α (eIF2α) kinase (PERK), and inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1). To investigate if this pathway is involved in the effects of23and26on BRD4 protein levels, we evaluated the expression ofAtf4, Atf6, and Xbp1s genes, as each of these transcription factors participates in one of the three branching pathways of the UPR activation.35 As shown in Figure 6B, both compounds could activate UPR, although through a mechanism different from that of thapsigargin (TG), a known ER stress inducer. In fact, while all 3 genes were induced by TG treatment, onlyAtf4increased (up to 5-fold compared to controls) upon exposure to compound23or compound26(Figure 6B)

Description

As the degradation induced by23(bearing the adamantane-based tag) and26(bearing methyldiphenylmethane) was significant under these conditions, these two compounds were selected for further analysis. We performed a dose−response curve (0.3−30 μM) for 8 h that allowed us to demonstrate the dose-dependency of both compounds in inducing such degradation (Figure 5B). Notably, compound26was more potent as it caused a more marked degradation of BRD4 (DC50 of 10.2 μM and Dmax of 83.3 %), compared to compound23(DC50 of 24.7 μM and Dmax of 57.4 %).

Description

As the degradation induced by23(bearing the adamantane-based tag) and26(bearing methyldiphenylmethane) was significant under these conditions, these two compounds were selected for further analysis. We performed a dose−response curve (0.3−30μM) for 8 h that allowed us to demonstrate the dose-dependency of both compounds in inducing such degradation (Figure 5B). Notably, compound26was more potent as it caused a more marked degradation of BRD4 (DC50of 10.2μM and Dmax of 83.3%), compared to compound23(DC50 of 24.7μM and Dmax of 57.4 %).

Description

BRD4 is widely recognized for regulating the expression of oncogenes in cancer cells. Therefore, we evaluated whether targeted inhibition of BRD4, following exposure to HyTags, could induce growth arrest or apoptosis of the 4T1 breast cancer cell line.36 As summarized in the graphs depicted in Figure 6C, both HyTags23and26caused a dose-dependent cytotoxicity, with an IC50 of 20.6±3.2 μM and 15.9±7.3 μM, respectively.