Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC50 values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.

Cancer immunotherapy, such as immune checkpoint blockade (ICB), has emerged as a groundbreaking approach for effective cancer treatment. Despite its considerable potential, clinical studies have indicated that the current response rate to cancer immunotherapy is suboptimal, primarily attributed to low immunogenicity in certain types of malignant tumors. Immunogenic cell death (ICD) represents a form of regulated cell death (RCD) capable of enhancing tumor immunogenicity and activating tumor-specific innate and adaptive immune responses in immunocompetent hosts. Therefore, gaining a deeper understanding of ICD and its evolution is crucial for developing more effective cancer therapeutic strategies. This review focuses exclusively on both historical and recent discoveries related to ICD modes and their mechanistic insights, particularly within the context of cancer immunotherapy. Our recent findings are also highlighted, revealing a mode of ICD induction facilitated by atypical interferon (IFN)-stimulated genes (ISGs), including polo-like kinase 2 (PLK2), during hyperactive type I IFN signaling. The review concludes by discussing the therapeutic potential of ICD, with special attention to its relevance in both preclinical and clinical settings within the field of cancer immunotherapy.

Synaptic plasticity is essential for memory encoding and stabilization of neural network activity. Plasticity is impaired in neurodegenerative conditions including Alzheimer disease (AD). A central factor in AD is amyloid precursor protein (APP). Previous studies have suggested APP involvement in synaptic plasticity, but physiological roles of APP are not well understood. Here, we identified combinatorial phosphorylation sites within APP that regulate AMPA receptor trafficking during different forms of synaptic plasticity. Dual phosphorylation sites at threonine-668/serine-675 of APP promoted endocytosis of the GluA2 subunit of AMPA receptors during homeostatic synaptic plasticity. APP was also required for GluA2 internalization during NMDA receptor-dependent long-term depression, albeit via a distinct pair of phosphoresidues at serine-655/threonine-686. These data implicate APP as a central gate for AMPA receptor internalization during distinct forms of plasticity, unlocked by specific combinations of phosphoresidues, and suggest that APP may serve broad functions in learning and memory.

The (poly)pharmacology of drug metabolites is seldom comprehensively characterized in drug discovery. However, some drug metabolites can reach high plasma concentrations and display in vivo activity. Here, we use computational and experimental methods to comprehensively characterize the kinase polypharmacology of M324, the major metabolite of the PARP1 inhibitor rucaparib. We demonstrate that M324 displays unique PLK2 inhibition at clinical concentrations. This kinase activity could have implications for the efficacy and safety of rucaparib and therefore warrants further clinical investigation. Importantly, we identify synergy between the drug and the metabolite in prostate cancer models and a complete reduction of α-synuclein accumulation in Parkinson\'s disease models. These activities could be harnessed in the clinic or open new drug discovery opportunities. The study reported here highlights the importance of characterizing the activity of drug metabolites to comprehensively understand drug response in the clinic and exploit our current drug arsenal in precision medicine.

BACKGROUND: Synucleinopathies, which are major pathological features of Parkinson\'s disease (PD), are characterized by misfolded aggregates of α-synuclein in the peripheral and central nervous system. Icariin (ICA) is the main active component of Epimedium flavonoids. Our previous study found that ICA decreases α-synuclein expression in APPV717I transgenic mice.
METHODS: The aim of the present study was to examine the potential applications and mechanisms of ICA in PD using A53T α-synuclein transgenic (A53T Tg) mice. After 3 months of intragastric ICA administration, rotarod and pole tests were used to assess behavioral changes in A53T Tg mice at 8 and 13 months of age. SH-SY5Y cells over-expressing wild-type α-synuclein were used to further examine the pharmacological effect and underlying mechanism of ICA. Western blotting and immunocytochemistry were used to detect the expression levels of α-synuclein and its related proteins.
RESULTS: ICA significantly improved the impaired motor function and coordination in A53T Tg mice. It also decreased the expression, Ser129 phosphorylation, and aggregation of α-synuclein in SH-SY5Y cells transfected with α-synuclein and the striatum of A53T Tg mice. Moreover, ICA increased the expression of parkin, which is associated with the ubiquitin-proteasome system (UPS), and decreased the level of polo-like kinase 2 (PLK2), an enzyme that phosphorylates α-synuclein.
CONCLUSIONS: ICA alleviated motor impairments in A53T mice, an effect which may be associated with the decreased phosphorylation and aggregation of α-synuclein through PLK2 and parkin regulation.

Synaptogenesis in the brain is highly organized and orchestrated by synaptic cellular adhesion molecules (CAMs) such as N-cadherin and amyloid precursor protein (APP) that contribute to the stabilization and structure of synapses. Although N-cadherin plays an integral role in synapse formation and synaptic plasticity, its function in synapse dismantling is not as well understood. Synapse weakening and loss are prominent features of neurodegenerative diseases, and can also be observed during homeostatic compensation to neuronal hyperexcitation. Previously, we have shown that during homeostatic synaptic plasticity, APP is a target for cleavage triggered by phosphorylation by Polo-like kinase 2 (Plk2). Here, we found that Plk2 directly phosphorylates N-cadherin, and during neuronal hyperexcitation Plk2 promotes N-cadherin proteolytic processing, degradation, and disruption of complexes with APP. We further examined the molecular mechanisms underlying N-cadherin degradation. Loss of N-cadherin adhesive function destabilizes excitatory synapses and promotes their structural dismantling as a prerequisite to eventual synapse elimination. This pathway, which may normally help to homeostatically restrain excitability, could also shed light on the dysregulated synapse loss that occurs in cognitive disorders.

Epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line therapy for EGFR mutated non-small cell lung cancer (NSCLC); however, resistance rapidly develops. The objective of this study was therefore to establish and characterize a gefitinib resistant NSCLC line (HCC827GR) and evaluate the therapeutic effects of natural products in combination with third-generation EGFR-TKI, AZD9291. The IC50 of gefitinib and AZD9291 in HCC827GR were significantly higher than those of HCC827 (p < 0.05). Furthermore, anchorage-independent colony assay indicated that HCC827GR cells were more aggressive than their predecessors. This was reflected by the gene/protein expression changes observed in HCC827GR versus HCC827 profiled by cancer drug resistance real-time polymerase chain reaction (RT-PCR) array and Western blot. Three natural products were screened and caffeic acid phenethyl ester (CAPE) exhibited the most significant combinative cytotoxic effect with AZD9291. Specifically, flow cytometry revealed that AZD9291 + CAPE considerably increased the fraction of cell in pre-G1 of the cell cycle and caspase-Glo3/7 assay showed a dramatic increase in apoptosis when compared to AZD9291 alone. Furthermore, Western blot showed significant downregulation of p-EGFR/p-AKT in HCC827GR cells treated with AZD9291 + CAPE as compared to AZD9291. Moreover, it is evident that AZD9291 + CAPE specifically resulted in a marked reduction in the protein expressions of the cell-proliferation-related genes p21, cyclin D1, and survivin. Finally, refined RT-PCR/Western blot data indicated that AZD9291 + CAPE may at least partially exert its synergistic effects via the PLK2 pathway. Together, these results suggest that CAPE is a clinically relevant compound to aid AZD9291 in treating EGFR-TKI resistant cells through modulating critical genes/proteins involved in cancer resistance/therapy.

Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress.

The Plk2 is a cellular stress-responsive factor that is induced in response to oxidative stress. However, the roles of Plk2 in acute kidney injury (AKI) have not been clarified. We previously found that Plk2 is an interacting factor of Nrf2 in response to cellular stress, since Plk2 is upregulated in the Nrf2-dependent network. Here, we show that the levels of p53, Plk2, p21cip1, and chromatin-bound Nrf2 were all upregulated in kidney tissues of mice or NRK52E cells treated with either cisplatin or methotrexate. Upregulation of Plk2 by p53 led to an increase of Nrf2 in both soluble and chromatin fractions in cisplatin-treated NRK52E cells. Consistently, depletion of Plk2 suppressed the levels of Nrf2. Of note, Plk2 directly phosphorylated Nrf2 at Ser40, which facilitated its interaction with p21cip1 and translocation into the nuclei for the activation of anti-oxidative and anti-inflammatory factors in response to AKI. Together, these findings suggest that Plk2 may serve as an anti-oxidative and anti-inflammatory regulator through the phosphorylation and activation of Nrf2 to protect kidney cells from kidney toxicants and that Plk2 and Nrf2 therefore work cooperatively for the protection and survival of kidney cells from harmful stresses.

Colorectal cancer (CRC) is a worldwide disease with worse survival. Our objective is to identify previously unrecognized prognostic factors to better evaluate disease progression. Seven GEO datasets were collected and analysed using R software, followed by KEGG enrichment analysis and TFs network construction. LASSO-COX analysis was performed to select the most useful prognostic features. COX model was used to analyse prognostic factors associated with OS. The survival curve was constructed using Kaplan-Meier analysis. A Nomogram model was also constructed to predict prognosis. A total of 3559 differentially expressed genes (DEGs) and 66 differentially expressed transcription factors were identified. FOXD1 was identified as the most differentially expressed factor of TFs covering the most downstream DEGs and independent risk prognostic factor. Next, FOXD1 expression was detected using immunohistochemical staining in 131 CRC patients\' tissue and the association between FOXD1 expression and clinicopathologic features was analysed. High expression of FOXD1 was correlated with TNM stage and pathological differentiation. Multivariate COX regression analyses confirmed that FOXD1 high-expression, TNM stage and tumour differentiation were independent prognostic risk factor of OS and DFS. Patients with high expression of FOXD1 were more likely to have poor overall survival and disease-free survival. The combination of FOXD1 and Plk2 which we have previously reported allowed us to predict the survival of post-surgical CRC patients more accurately, adding to the former prognostic model based on the TNM Stage. The results showed that patients with high expression of both FOXD1 and Plk2 have the worst survival. A combination of FOXD1 and Plk2 can better evaluate patients\' survival.