CZY43

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

To explore the mechanism of HER3 degradation by compound CZY43, we conducted co-immunoprecipitation experiment to investigate the interaction of HER3 with Hsp90, which is a molecular chaperone protein for sensing correct protein folding [25]. As shown in Fig. 4A, treatment of compound CZY43 increased the binding of Hsp90 to HER3. Additionally, the ubiquitination levels of HER3 was also increased (Fig. 4A). Further exploration revealed that the degradation of HER3 by compound CZY43 can be rescued by the autophagy inhibitor chloroquine (CQ) but not the proteasome inhibitor MG132, indicating that compound CZY43-induced HER3 degradation might went through the autophagic pathway (Fig. 4B and C). To further verify that compound CZY43 induced HER3 degradation through autophagy, we detected the activated version of autophagy-related protein LC3B using western blot. The result showed that treatment with compound CZY43 increased the level of LC3B II, an active form of LC3B that appears when autophagy happens (Fig. 4D) [26]. These results indicated that compound CZY43 induced HER3 degradation via autophagy.

Description

To further characterize compound CZY43, we treated SKBR3 cells with increasing concentrations of this compound for 8 h. It was shown that compound CZY43 dose-dependently induced degradation of HER3 protein(Fig. 3A and B).HER3 degradation rate at the indicated concentration of compound CZY43 was plotted for DC50 determination. Protein levels were quantified using ImageJ and normalized to corresponding actin, then plotted using Graphpad Prism 8.0. The data were representative of three independent experiments. Error bars showed mean ± SD.

Description

We also evaluated the anti-proliferative effect of compound CZY43 on SKBR3 cells. The result showed that compound CZY43 potently inhibited SKBR3 cell growth with an IC50 value of 0.53 μ M, which is much lower than that of compound 4 (5.14 μ M) (Fig. 6A). Additionally, we also observed that compound CZY43 effectively inhibited the adhesion of SKBR3 cells (Fig. 6B and C). These results indicated that compound CZY43 effectively inhibited cancer cell growth and adhesion.