LPM3770277

Amino Acid Sequence

3D Structure (/structures/AF-P11802-F1-model_v6.cif)

Function

Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G1/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes and the subsequent transcription of E2F target genes which are responsible for the progression through the G1 phase. Hypophosphorylates RB1 in early G1 phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex.

Amino Acid Sequence

3D Structure (/structures/AF-Q00534-F1-model_v6.cif)

Function

Serine/threonine-protein kinase involved in the control of the cell cycle and differentiation; promotes G1/S transition. Phosphorylates pRB/RB1 and NPM1. Interacts with D-type G1 cyclins during interphase at G1 to form a pRB/RB1 kinase and controls the entrance into the cell cycle. Involved in initiation and maintenance of cell cycle exit during cell differentiation; prevents cell proliferation and negatively regulates cell differentiation, but is required for the proliferation of specific cell types (e.g. erythroid and hematopoietic cells). Essential for cell proliferation within the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles. Required during thymocyte development. Promotes the production of newborn neurons, probably by modulating G1 length. Promotes, at least in astrocytes, changes in patterns of gene expression, changes in the actin cytoskeleton including loss of stress fibers, and enhanced motility during cell differentiation. Prevents myeloid differentiation by interfering with RUNX1 and reducing its transcription transactivation activity, but promotes proliferation of normal myeloid progenitors. Delays senescence. Promotes the proliferation of beta-cells in pancreatic islets of Langerhans. May play a role in the centrosome organization during the cell cycle phases.

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

What was equally surprising was that after inhibiting lysosomes by bafilomycin A1, the cytotoxic effect of LPM3770277 was significantly reduced (Figure 5A). The use of another lysosome inhibitor, NH4CL, led to similar results (Supplementary Figure S2B). To further solve this puzzle, we extracted the lysosomes of HCC1806 cells after drug treatment, and used HPLC to detect the drug content in the lysosome. The result showed that under the same condition, the drug concentration of LPM3770277 in the lysosome was 24 times higher than that of abemaciclib (Figure 5B). This suggests that LPM3770277 might operate via certain lysosome-dependent functions such as autophagy. To test this hypothesis, we examined the effects of abemaciclib and LPM3770277 treatments on autophagy-related proteins in HCC1806 cells. Figure 5C showed that LPM3770277 treatment increased the expression of LC3B protein and decreased the expression of P62 protein, both of which are crucial players in autophagy. These results indicate that LPM3770277 could significantly increase the autophagy level in HCC1806 cells, and that LPM3770277 may degrade CDK4/6 protein through lysosomal-mediated autophagy.

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 used a proteasome inhibitor bortezomib to block proteasome activity and it was found that bortezomib (10 nM) significantly and almost completely prevented the CDK4/6-degradating activity of LPM3770277 (Figure 4) . This result suggested that LPM3770277 degraded CDK4/6 protein via proteasome to exert its anti-cancer cell proliferation activity

Description

Binding experiment showed that LPM3770277 had a slightly weaker binding affinity on CDK4kinase, which may be due to the hydrophobic tags on LPM3770277 which could increase its steric hindrance (Supplementary Figure S1). Figure S1: Binding activity of abemaciclib,LPM3770277and LPM3770304 to CDK4 and CvclinD3 and its IC50.