Rational Molecular Design of Potent PLK1 PBD Domain-binding Phosphopeptides Using Preferential Amino Acid Building Blocks.

Polo-like kinase 1 (PLK1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo-box domain (PBD) present in C-terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD-substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain-peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD-binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD-substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6-mer phosphopeptides (i.e., IQSpSPC, LQSpTPF, and LNSpTPT) were successfully developed, which can efficiently target PBD domain with high affinity (Kd = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 µm, resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK1 PBD domain with a newly designed, potent phosphopeptide LQSpTPF as well as diverse noncovalent chemical forces, such as H-bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.

Aß peptide secretion is reduced by Radix Polygalae-induced autophagy via activation of the AMPK/mTOR pathway.

Radix Polygalae is a traditional Chinese medicine that has been used as a sedative and to improve memory for a number of years. The impact of Radix Polygalae in patients with Alzheimer's disease has been investigated. However the mechanisms underlying its effects remain unclear. In the current study, the toxicity of various doses (100, 40, 20, 10, 5 and 0 µg/ml) of Radix Polygalae was measured in the human neuroblastoma cell line (SH-SY5Y) using an MTT assay. Changes in amyloid ß (Aß) levels in the supernatant of Chinese hamster ovary (CHO) cells overexpressing ß-amyloid pro-protein (APP) and BACE1 (CHO-APP/BACE1), were detected using an ELISA assay. In order to confirm that the Aß reduction was associated with autophagy, the autophagy marker protein, light chain 3 (LC3), was measured by western blot analysis and autophagosomes were assessed using MDC staining. In addition, the mechanism underlying the autophagy induced by Radix Polygalae was analyzed using western blotting to measure the protein expression of mammalian target of rapamycin (mTOR), p70s6k, Raptor, protein kinase B and adenosine monophosphate-activated protein kinase (AMPK), in addition to the phosphorylated forms of these proteins. The results demonstrated no significant toxicity of Radix Polygalae in SH-SY5Y cells, at a dose of 100 µg/ml. The secretion of Aß was markedly reduced following treatment with Radix Polygalae, and this reduction occurred in a dose-dependent manner. The autophagy levels were shown to be enhanced in the drug treatment group, using fluorescence microscopy. In addition, levels of LC3II/LC3I, the marker protein of autophagy, were also increased. The results of the current study suggest that Radix Polygalae may aid in the elimination of the Aß peptide, via the induction of autophagy, by the AMPK/mTOR signaling pathway. These results may provide a basis for further kin vivo investigation.