HyT-S7

Amino Acid Sequence

3D Structure (/structures/AF-P21860-F1-model_v6.cif)

Function

Tyrosine-protein kinase that plays an essential role as cell surface receptor for neuregulins. Binds to neuregulin-1 (NRG1) and is activated by it; ligand-binding increases phosphorylation on tyrosine residues and promotes its association with the p85 subunit of phosphatidylinositol 3-kinase. May also be activated by CSPG5. Involved in the regulation of myeloid cell differentiation.

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

Firstly, to confirm whether the ubiquitin-proteasome system is involved in the degradation of HBC induced by HyT-S7, we investigated the effect of proteasome inhibitors on the degradation efficacy of HyT-S7. As shown in Fig. 6A, MG132, and bortezomib failed to reverse the degradation effect of HyT-S7 on HBC. Subsequently, as illustrated in Fig. 6B, the addition of 17-AAG did not affect the degradation of HBC by HyT-S7. These experimental findings suggest that HyT-S7 does not induce the degradation of HBC through the ubiquitin-proteasome system mediated by Hsp70. Next, we co-treated HepAD38 cells with autophagy inhibitors and HyT-S7, and the results were illustrated in Fig. 6C. Both 3-MA and HCQ could reverse the degradation effect of HyTS7 on HBC. Additionally, it has been reported that MG132 could act as an autophagy activator, which can be observed from Fig. 6A that the addition of MG132 promotes the degradation of HBC induced by HyT-S7. Furthermore, a data-independent acquisition (DIA)-based proteomic analysis was performed to evaluate differentially expressed proteins on HepAD38 cells. Treated with HyT-S7. In total, 8013 proteins were identified and quantified from our proteomic data. Based on fold-change screening and comparison with the DMSO group, we identified 234 upregulated proteins and 224 downregulated proteins (fold change >1.2 or <0.83, P < 0.05) after HyT-S7 treatment. We did observe a significant increase or decrease in proteins involved in the autophagy pathway, such as JUN, SQSTM1 (p62), LC3B, WIPI1, TBC1D17, ERN1, TMEM39A, TMEM41B and SKP2 (Fig. 6D). Moreover, the KEGG pathways analysis showed that there are 33 proteins related to transport, catabolism, fording, sorting and degradation underwent significant systemic changes in the presence of HyT-S7 (Fig. 6E). Collectively, these results indicate that HyT-S7 can induce the degradation of HBC through the autophagy pathway.

Description

To further evaluate the degradation efficiency of HyT-S7, HepAD38 cells were treated with gradient-diluted HyT-S7, followed by cell lysis 24 h later and Western blot assay. The results of three independent replicate experiments were selected for quantitative analysis. As depicted in Fig.5A and B, HyT-S7 could degrade HBC in a concentration-dependent manner. Based on the quantified results, the half-maximal degradation concentration(DC50)of HyTS7 for HBC was calculated to be 3 .02± 0.54 μmol/L.

Description

However, when PEG was employed as a linker with a carbon atom number of seven, HyT-S7 not only demonstrated good antiviral activity (EC50 =0.46μmol/L) but also significantly reduced cytotoxicity (CC50 =28.84 μmol/L), making it a promising candidate for further modification as an advantageous linker. EC50 is the concentration of the test compounds for reduction of HBV DNA by 50% in HepAD38 cells. EC50 values were evaluated from HBV DNA by the qPCR assay after 6 days of incubating the cells with the compounds.

Description

when PEG was employed as a linker with a carbon atom number of seven, HyT-S7 not only demonstrated good antiviral activity (EC50 =0.46 μmol/L) but also significantly reduced cytotoxicity (CC50 =28.84 μmol/L), making it a promising candidate for further modification as an advantageous linker. CC50 is the concentration of the test compounds for reduction of the viability of HepAD38 cells by 50%. CC50 values were measured by the CPE method.