Application of a Fluorescence Recovery-Based Polo-Like Kinase 1 Binding Assay to Polo-Like Kinase 2 and Polo-Like Kinase 3.

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.

Emerging role of immunogenic cell death in cancer immunotherapy.

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.

Amyloid precursor protein combinatorial phosphorylation code regulates AMPA receptor removal during distinct forms of synaptic plasticity.

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.

Identification of differential biological activity and synergy between the PARP inhibitor rucaparib and its major metabolite.

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.

Plk2 promotes synaptic destabilization through disruption of N-cadherin adhesion complexes during homeostatic adaptation to hyperexcitation.

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.

Caffeic acid phenethyl ester surmounts acquired resistance of AZD9291 in non-small cell lung cancer cells.

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.

Plk2-mediated phosphorylation and translocalization of Nrf2 activates anti-inflammation through p53/Plk2/p21cip1 signaling in acute kidney injury.

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.

Icariin Inhibits Overexpression and Aggregation of α-Synuclein in A53T α-Synuclein Transgenic Mice by Regulating Parkin and PLK2.

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.

Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5.

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.

Combination of FOXD1 and Plk2: A novel biomarker for predicting unfavourable prognosis of colorectal cancer.

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.

Circular RNA 0102049 suppresses the progression of osteosarcoma through modulating miR-520g-3p/PLK2 axis.

Circular RNAs (circRNAs) are a type of non-coding RNAs generated from back splicing to enhance or inhibit the progression of multiple human cancers including osteosarcoma (OS). Although circ_0102049 has been found to be highly expressed in OS cell lines, the role and specific mechanism of circ_0102049 in OS remains unclear. Here, we found that silence of circ_0102049 could significantly exacerbate the tumorigenesis of OS in vivo through sponging microRNA-520g-3p. Polo-like kinase 2 (PLK2) was predicted to be a target of miR-520g-3p, and luciferase reporter assay revealed that overexpression of miR-520g-3p dramatically suppressed the expression of PLK2, whereas miR-520g-3p inhibitor promoted the PLK2 expression. Moreover, the silence of circ_0102049 could markedly promote the proliferation, invasion, migration and cell-cycle promotion while inhibiting the apoptosis of OS cell line MG63 cells in vitro through regulating miR-520g-3p/PLK2 axis. Taken together, the present study indicated that circ_0102049 suppressed the progression of osteosarcoma via modulating miR-520g-3p/PLK2/TAp73 axis, providing a potential therapeutic target for OS.

PLK2 protects retinal ganglion cells from oxidative stress by potentiating Nrf2 signaling via GSK-3ß.

Oxidative stress of retinal ganglion cells (RGCs) has been established as a main contributor to retinal degeneration in the pathogenesis of glaucoma. Polo-like kinase 2 (PLK2) has recently been reported to be a potent antioxidant protein that enhances cell survival in response to oxidative stress. To date, the involvement of PLK2 in RGC-associated oxidative stress is undermined. In the present work, we evaluated whether PLK2 regulates oxidative stress evoked by hydrogen peroxide (H2 O2 ) in RGCs. PLK2 expression was induced by H2 O2 stimulation in RGCs. Upregulation of PLK2 had a profoundly cytoprotective effect on H2 O2 -stimulated RGCs by attenuating cellular apoptosis and reactive oxygen species (ROS) level. Further data revealed that upregulation of PLK2 strikingly enhanced the activation of Nrf2 signaling. Moreover, PLK2 overexpression promoted glycogen synthase kinase (GSK)-3ß phosphorylation, whereas PLK2 knockdown reduced the levels of GSK-3ß phosphorylation. Notably, GSK-3ß inhibition using a chemical inhibitor markedly abrogated the suppressive effects of PLK2 knockdown on Nrf2 activation. Repression of Nrf2 blocked the PLK2 overexpression-induced protective effects in H2 O2 -stimulated RGCs. Overall, this study elucidates that upregulation of PLK2 protects RGCs against H2 O2 -induced oxidative stress injury by upregulating Nrf2 activation via modulation of GSK-3ß phosphorylation. These findings underline the pivotal role of PLK2 in mediating oxidative stress-evoked retinal degeneration in the pathogenesis of glaucoma.

Genetic Deletion of Polo-Like Kinase 2 Induces a Pro-Fibrotic Pulmonary Phenotype.

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.

Knocking down of Polo-like kinase 2 inhibits cell proliferation and induced cell apoptosis in human glioma cells.

AIMS: Polo-like kinase 2 (PLK2) belongs to a family of serine/threonine kinases, and it is involved in tumorigenesis. The present study aimed to explore the potential clinical significance of PLK2 in the development of gliomas. MAIN METHODS: Immunohistochemistry (IHC) was performed to detect the expression of PLK2 in glioma tissues. Cell proliferation and apoptosis were determined by Cell Counting Kit 8 (CCK8) and flow cytometry analysis, respectively. KEY FINDINGS: PLK2 expression gradually increased with the degree of glioma malignancy. High PLK2 expression was associated with a poor prognosis in glioma. Short hairpin RNAs targeting PLK2 (shPLK2) inhibited the viability and induced apoptosis of glioma cells, both in vitro and in vivo. Ring finger protein 180 (RNF180), an E3 ubiquitin ligase, interacted with PLK2 and induced the ubiquitination of PLK2. Overexpression of PLK2 in glioma cells significantly inhibited RNF180 upregulation-induced cell apoptosis. The expression level of RNF180 gradually decreased with the degree of glioma malignancy. SIGNIFICANCE: Knocking down of PLK2 may suppress the glioma development through cancer cell proliferation inhibition and cell apoptosis promotion. Furthermore, RNF180 may mediate the ubiquitination of PLK2. The present findings may help improve the clinical management of glioma in the future.

Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling.

BACKGROUND: Glioblastoma (GBM) is a lethal type of primary brain tumor with a median survival less than 15 months. Despite the recent improvements of comprehensive strategies, the outcomes for GBM patients remain dismal. Accumulating evidence indicates that rapid acquired chemoresistance is the major cause of GBM recurrence thus leads to worse clinical outcomes. Therefore, developing novel biomarkers and therapeutic targets for chemoresistant GBM is crucial for long-term cures. METHODS: Transcriptomic profiles of glioblastoma were downloaded from gene expression omnibus (GEO) and TCGA database. Differentially expressed genes were analyzed and candidate gene PLK2 was selected for subsequent validation. Clinical samples and corresponding data were collected from our center and measured using immunohistochemistry analysis. Lentiviral transduction and in vivo xenograft transplantation were used to validate the bioinformatic findings. GSEA analyses were conducted to identify potential signaling pathways related to PLK2 expression and further confirmed by in vitro mechanistic assays. RESULTS: In this study, we identified PLK2 as an extremely suppressed kinase-encoding gene in GBM samples, particularly in therapy resistant GBM. Additionally, reduced PLK2 expression implied poor prognosis and TMZ resistance in GBM patients. Functionally, up-regulated PLK2 attenuated cell proliferation, migration, invasion, and tumorigenesis of GBM cells. Besides, exogenous overexpression of PLK2 reduced acquired TMZ resistance of GBM cells. Furthermore, bioinformatics analysis indicated that PLK2 was negatively correlated with Notch signaling pathway in GBM. Mechanically, loss of PLK2 activated Notch pathway through negative transcriptional regulation of HES1 and degradation of Notch1. CONCLUSION: Loss of PLK2 enhances aggressive biological behavior of GBM through activation of Notch signaling, indicating that PLK2 could be a prognostic biomarker and potential therapeutic target for chemoresistant GBM.

Targeting Adaptive IRE1α Signaling and PLK2 in Multiple Myeloma: Possible Anti-Tumor Mechanisms of KIRA8 and Nilotinib.

BACKGROUND: Inositol-requiring enzyme 1α (IRE1α), along with protein kinase R-like endoplasmic reticulum kinase (PERK), is a principal regulator of the unfolded protein response (UPR). Recently, the 'mono'-specific IRE1α inhibitor, kinase-inhibiting RNase attenuator 6 (KIRA6), demonstrated a promising effect against multiple myeloma (MM). Side-stepping the clinical translation, a detailed UPR phenotype in patients with MM and the mechanisms of how KIRA8 works in MM remains unclear. METHODS: We characterized UPR phenotypes in the bone marrow of patients with newly diagnosed MM. Then, in human MM cells we analyzed the possible anti-tumor mechanisms of KIRA8 and a Food and Drug Administration (FDA)-approved drug, nilotinib, which we recently identified as having a strong inhibitory effect against IRE1α activity. Finally, we performed an RNA-sequence analysis to detect key IRE1α-related molecules against MM. RESULTS: We illustrated the dominant induction of adaptive UPR markers under IRE1α over the PERK pathway in patients with MM. In human MM cells, KIRA8 decreased cell viability and induced apoptosis, along with the induction of C/EBP homologous protein (CHOP); its combination with bortezomib exhibited more anti-myeloma effects than KIRA8 alone. Nilotinib exerted a similar effect compared with KIRA8. RNA-sequencing identified Polo-like kinase 2 (PLK2) as a KIRA8-suppressed gene. Specifically, the IRE1α overexpression induced PLK2 expression, which was decreased by KIRA8. KIRA8 and PLK2 inhibition exerted anti-myeloma effects with apoptosis induction and the regulation of cell proliferation. Finally, PLK2 was pathologically confirmed to be highly expressed in patients with MM. CONCLUSION: Dominant activation of adaptive IRE1α was established in patients with MM. Both KIRA8 and nilotinib exhibited anti-myeloma effects, which were enhanced by bortezomib. Adaptive IRE1α signaling and PLK2 could be potential therapeutic targets and biomarkers in MM.

PLK2 modulation of enriched TAp73 affects osteogenic differentiation and prognosis in human osteosarcoma.

There are three subtypes of undifferentiated human conventional osteosarcoma (HCOS): osteoblastic osteosarcoma (OOS), chondroblastic osteosarcoma (COS), and fibroblastic osteosarcoma (FOS). HCOS also exhibits heterogeneous pathological maldifferentiation in individual patients. Currently, the mechanism regulating HCOS differentiation remains unclear, and therapies are ineffective. Osteopontin (OPN) and osteocalcin (OCN) are markers of osteoblast maturation, and their expression is inhibited in HCOS. A previous study found that PLK2 inhibited TAp73 phosphorylation and consequent anti-OS function of TAp73 in OS cells with enriched TAp73. TAp73 was also reported to regulate bone cell calcification. Here, OOS was found to have higher TAp73 levels and PLK2 expression than those in COS, which is correlated with HCOS maldifferentiation according to Spearman analysis and affects patient prognosis according to Kaplan-Meier survival analysis. In the conventional OS cell-line Saos2 and in patient-derived xenograft OS (PDX-OS) cells, increased PLK2 expression owing to abundant TAp73 levels affected OPN and OCN content as measured by RT-PCR and Western blotting, and alizarin red staining showed that PLK2 affected calcium deposition in OS cells. In addition, PLK2 inhibition in PDX-OS cells prohibited clone formation, as indicated by a clonogenic assay, and sensitized OS cells to cisplatin (CDDP) (which consequently limited proliferation), as shown by the CCK-8 assay. In an established PDX animal model with abundant TAp73 levels, PLK2 inhibition or CDDP treatment prevented tumor growth and prolonged median survival. The combined therapeutic effect of PLK2 inhibition with CDDP treatment was better than that of either monotherapy. These results indicate that increased PLK2 levels due to enriched TAp73 affect osteogenic differentiation and maturation and OS prognosis. In conclusion, PLK2 is a potential target for differentiation therapy of OS with enriched TAp73.

Proteome Analysis in a Mammalian Cell line Reveals that PLK2 is Involved in Avian Metapneumovirus Type C (aMPV/C)-Induced Apoptosis.

Avian metapneumovirus subtype C (aMPV/C) causes an acute respiratory disease that has caused serious economic losses in the Chinese poultry industry. In the present study, we first explored the protein profile in aMPV/C-infected Vero cells using iTRAQ quantitative proteomics. A total of 921 of 7034 proteins were identified as significantly altered by aMPV/C infection. Three selected proteins were confirmed by Western blot analysis. Bioinformatics GO analysis revealed multiple signaling pathways involving cell cycle, endocytosis, and PI3K-Akt, mTOR, MAPK and p53 signaling pathways, which might participate in viral infection. In this analysis, we found that PLK2 expression was upregulated by aMPV/C infection and investigated whether it contributed to aMPV/C-mediated cellular dysfunction. Suppressing PLK2 attenuated aMPV/C-induced reactive oxygen species (ROS) production and p53-dependent apoptosis and reduced virus release. These results in a mammalian cell line suggest that high PLK2 expression correlates with aMPV/C-induced apoptosis and viral replication, providing new insight into the potential avian host cellular response to aMPV/C infection and antiviral targets.

Insulin Resistance Promotes Parkinson's Disease through Aberrant Expression of α-Synuclein, Mitochondrial Dysfunction, and Deregulation of the Polo-Like Kinase 2 Signaling.

Background: Insulin resistance (IR), considered a hallmark of diabetes at the cellular level, is implicated in pre-diabetes, results in type 2 diabetes, and negatively affects mitochondrial function. Diabetes is increasingly associated with enhanced risk of developing Parkinson's disease (PD); however, the underlying mechanism remains unclear. This study investigated the probable culpability of IR in the pathogenesis of PD. Methods: Using MitoPark mice in vivo models, diabetes was induced by a high-fat diet in the in vivo models, and IR was induced by protracted pulse-stimulation with 100 nM insulin treatment of neuronal cells, in vitro to determine the molecular mechanism(s) underlying altered cellular functions in PD, including mitochondrial dysfunction and α-synuclein (SNCA) aberrant expression. Findings: We observed increased SNCA expression in the dopaminergic (DA) neurons of both the wild-type and diabetic MitoPark mice, coupled with enhanced degeneration of DA neurons in the diabetic MitoPark mice. Ex vivo, in differentiated human DA neurons, IR was associated with increased SNCA and reactive oxygen species (ROS) levels, as well as mitochondrial depolarization. Moreover, we demonstrated concomitant hyperactivation of polo-like kinase-2 (PLK2), and upregulated p-SNCA (Ser129) and proteinase K-resistant SNCA proteins level in IR SH-SY5Y cells, however the inhibition of PLK2 reversed IR-related increases in phosphorylated and total SNCA. Similarly, the overexpression of peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC)-1α suppressed ROS production, repressed PLK2 hyperactivity, and resulted in downregulation of total and Ser129-phosphorylated SNCA in the IR SH-SY5Y cells. Conclusions: These findings demonstrate that IR-associated diabetes promotes the development and progression of PD through PLK2-mediated mitochondrial dysfunction, upregulated ROS production, and enhanced SNCA signaling, suggesting the therapeutic targetability of PLK2 and/or SNCA as potential novel disease-modifying strategies in patients with PD.

Plk2 Regulated by miR-128 Induces Ischemia-Reperfusion Injury in Cardiac Cells.

Ischemia-reperfusion (I/R) injury occurs during cardiac surgery and is the major factor leading to heart dysfunction and heart failure. Our previous study showed that gene and microRNA expression profiles are altered in heart grafts with extended I/R injury. In this study, we, for the first time, demonstrated that I/R injury upregulates the expression of Polo-like kinase 2 (Plk2) but decreases miR-128 expression in heart cells both in vitro and in vivo. Silencing Plk2 using small interfering RNA (siRNA) protects cells from Antimycin A-induced cell apoptosis/death. Silencing Plk2 also decreases phosphorylated p65 expression but increases Angiopoietin 1 expression. In addition, Plk2 is negatively regulated by miR-128. miR-128 exerts a protective effect on cell apoptosis similar to Plk2 siRNA in response to I/R stress. Methylation inhibitor 5-azacytidine (5-AZ) increases the expression of miR-128 and subsequently reduces Plk2 expression and cell apoptosis. In conclusion, this study demonstrated that Plk2 regulated by miR-128 induces cell apoptosis/death in response to I/R stress through activation of the nuclear factor κB (NF-κB) signal pathway. miR-128 and Plk2 are new targets for preventing cardiac I/R injury or oxidative stress-mediated injury.