Subunit Of Kinase Research Articles

Silencing arginine kinase/integrin β1 subunit by transgenic plant expressing dsRNA inhibits the development and survival of Plutella xylostella.

Plutella xylostella is a devastating agricultural insect pest of cruciferous plants, including crops. Plant-mediated RNA interference (RNAi) is currently being developed for plant protection. In this study, we investigated the response of P. xylostella exposed to transgenic Arabidopsis thaliana plants that expressed double-stranded RNA (dsRNA) targeting P. xylostella genes of arginine kinase (PxAK) and integrin β1 subunit (Pxβ). Transgenic plants producing dsRNAs of the 384-bp fragment of PxAK (dsAK plants), the 497-bp fragment of Pxβ (dsβ plants), and the 881 bp of the combination of both genes (dsAK-β plants) were generated and verified. Insect bioassay with these transgenic plants showed that the development of P. xylostella was affected, causing longer developmental time, and lower pupal weight and pupation rate. P. xylostella mortality rates were 25.0% when exposed to dsAK plants, 22.5% with dsβ plants, and 30.0% with dsAK-β plants, which were all higher than 7.5% for the wild-type plant. PxAK and Pxβ in P. xylostella were suppressed by 26.6-79.7% at the transcription level by the transgenic plants. These results suggest that plant-mediated RNAi targeting single gene or both PxAK and Pxβ may have the potential to control P. xylostella. © 2019 Society of Chemical Industry.

IKBKE enhances TMZ-chemoresistance through upregulation of MGMT expression in glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant type of brain tumor. Despite advances in diagnosis and therapy, the prognosis of patients with GBM has remained dismal. Multidrug resistance and high recurrence are two of the major challenges in successfully treating brain tumors. IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) is a major oncogenic protein in tumors and can inhibit glioblastoma cell proliferation, migration, and tumorigenesis. Our study aimed to investigate the mechanism of IKBKE enhancing the resistance of glioma cells to temozolomide. For the in vitro experiments, LN18 and U118 glioblastoma cells were treated with a combination of sh/oe-IKBKE lentivirus and TMZ. Cell proliferation was determined by the EdU assay and colony formation assays. Apoptosis was analyzed by the TUNEL assay. In vivo, LN18 NC and LN18 sh-IKBKE cells were implanted into the cerebrums of nude mice to detect the effect of combination therapy. The protein and mRNA levels were assayed by western blot, immunohistochemistry, and qRT-PCR. In this study, we demonstrated that IKBKE enhances the resistance of glioblastoma cells to temozolomide (TMZ) by activating the AKT/NF-κB signaling pathway to upregulate the expression of the DNA repair enzyme o6-methylguanine-dna methyltransferase (MGMT). In glioblastoma cells, IKBKE knockdown enhances apoptosis and suppresses cell proliferation, clone formation, and tumor development in vivo induced by TMZ. However, overexpression of IKBKE reduces the effects of TMZ. Our studies suggest that inhibition of IKBKE can enhance the therapeutic effect of TMZ on GBM in vitro and in vivo, providing new research directions and therapeutic targets for the treatment of GBM.

Vanillin derivative VND3207 activates DNA-PKcs conferring protection against radiation-induced intestinal epithelial cells injury in vitro and in vivo

Vanillin is a natural compound endowed with antioxidant and anti-mutagenic properties. We previously identified the vanillin derivative VND3207 with strong radio-protective and antioxidant effects and found that VND3207 confers survival benefit and protection against radiation-induced intestinal injury (RIII) in mice. We also observed that VND3207 treatment enhanced the expression level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in human lymphoblastoid cells with or without γ-irradiation. DNA-PKcs is a critical component of DNA double strand break repair pathway and also regulates mitotic progression by stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. In the present study, we found that VND3207 protected intestinal epithelial cells in vitro against ionizing radiation by promoting cell proliferation and inhibiting cell apoptosis. In addition, VND3207 promoted DNA-PKcs activity by increasing autophosphorylation at S2056 site. Consistent with this, VND3207 significantly decreased the number of γH2AX foci and mitotic catastrophe after radiation. DNA-PKcs deficiency abolished these VND3207 radio-protective effects, indicating that DNA-PKcs activation is essential for VND3207 activity. In conclusion, VND3207 promoted intestinal repair following radiation injury by regulating the DNA-PKcs pathway.

Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry.

Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.

Germline de novo variants in CSNK2B in Chinese patients with epilepsy

CSNK2B, which encodes the beta subunit of casein kinase II (CK2), plays an important role in neuron morphology and synaptic transmission. Variants in CSNK2B associated with epilepsy and/or intellectual disability (ID)/developmental delay (DD) have been reported in five cases only. Among the 816 probands suspected hereditary epilepsy whose initial report of trio-based whole exome sequencing (WES) were negative, 10 de novo pathogenic or likely pathogenic variants of CSNK2B in nine probands were identified after reanalysis of their raw Trio-WES data. Six of the nine epileptic patients had ID/DD. The age of seizure onset of these nine patients with CSNK2B variants ranged from 2–12 months. Eight patients had age of seizure onset of less than 6 months. The epilepsy of most probands (8/9) was generalized tonic-clonic seizure and clustered (6/9). Most patients had normal electroencephalogram (5/9) and brain magnetic resonance image (7/9) results. Most patients (7/9) had easy-to-control seizures. Levetiracetam was the most commonly used drug in seizure-free patients (5/7). The variants detected in five patients (5/9, 55.6%) were located in the zinc-binding domain. In summary, our research provided evidence that variants in CSNK2B are associated with epilepsy with or without ID/DD. CSNK2B-related epilepsy is relatively easy to be controlled. The zinc-binding domain appears to be the hotspot region for mutation.

Effects of Brain Irradiation in Immune-Competent and Immune-Compromised Mouse Models.

Patient-derived orthotopic xenografts (PDOXs) closely recapitulate primary human glioblastoma (GBM) tumors in terms of histology and genotype. Compared to other mouse strains, NOD-scid IL2Rgammanull (NSG) mice show excellent tumor take rates, which makes them an ideal host for PDOXs. However, NSG mice harbor a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), which renders them relatively radiosensitive. This has been a frequently voiced concern in studies involving ionizing radiation. In this study, we assessed brain toxicity in NSG mice compared to three other different mouse strains frequently used in radiation studies at radiation doses commonly used in experimental combination therapy studies. C3H/Sed/Kam, C57Bl/6, nude and NOD-scid IL2Rgammanull mice received a single dose of 4 Gy to the right brain hemispheres using an image-guided small animal irradiator. Brains were stained using H&E, luxol fast blue, and antibodies against IBA1 and GFAP one, two, four or six months postirradiation. Additional animals of all four strains were exposed to five daily fractions of 2 Gy (5 × 2 Gy), and tissue sections were stained 72 h later against gH2AX, NeuN, GFAP and IBA1. None of the mouse strains displayed radiation-induced toxicity at any of the time points tested. Radiation doses relevant for testing combination therapies can be safely applied to the brains of NSG mice without the occurrence of radiation-induced normal tissue toxicity.

Fat-lowering effects of isorhamnetin are via NHR-49-dependent pathway in Caenorhabditis elegans

Isorhamnetin (3-O-methylquercetin), a flavonol found in dill weed, sea buckthorn berries, kale and onions, has been suggested to have anti-obesity effects, but there is limited evidence of its mechanisms of action on lipid metabolism. The goal of this study was to investigate the effects of isorhamnetin on lipid metabolism using Caenorhabditis elegans as an animal model. Isorhamnetin reduced fat accumulation without affecting food intake or energy expenditure in C. elegans. The isorhamnetin's fat-lowering effects were dependent on nhr-49, a homolog of the human peroxisome proliferator-activated receptor alpha (PPARα). Isorhamnetin upregulated an enoyl-CoA hydratase (ech-1.1, involved in fatty acid β-oxidation) and adipose triglyceride lipase (atgl-1, involved in lipolysis) via NHR-49-dependent pathway at transcriptional levels. Isorhamnetin also upregulated the C. elegans AMP-activated protein kinase (AMPK) subunits homologs (aak-1 and aak-2), involved in energy homeostasis. These results suggest that isorhamnetin reduces body fat by increasing fat oxidation in part via NHR-49/PPARα-dependent pathway.

Advances in IKBKE as a potential target for cancer therapy.

IKBKE (inhibitor of nuclear factor kappa‐B kinase subunit epsilon), a member of the nonclassical IKK family, plays an important role in the regulation of inflammatory reactions, activation and proliferation of immune cells, and metabolic diseases. Recent studies have demonstrated that IKBKE plays a crucial regulatory role in malignant tumor development. In recent years, IKBKE, an important oncoprotein in several kinds of tumors, has been widely found to regulate a variety of cytokines and signaling pathways. IKBKE promotes the growth, proliferation, invasion, and drug resistance of various cancers. This paper makes a detailed review that focuses on the recent discoveries of IKBKE in the malignant tumors, and puts forward that IKBKE is becoming an important therapeutic target for clinical treatment, which has been more and more realized.

Biochemical characterization of the cAMP-dependent protein kinase regulatory subunit-like protein from Trypanosoma equiperdum, detection of its inhibitory activity, and identification of potential interacting proteins

An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5–20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T. equiperdum PKA regulatory subunit-like protein (TeqR-like) on the basis of Western blot and mass spectrometry analyses, and behaved as an uncompetitive or anti-competitive inhibitor of the parasite TeqC-like protein, with respect to a fluorescently labeled substrate (kemptide, sequence: LRRASLG), showing a Ki of 1.17 μM. The isolated protein possesses a molecular mass of 57.54 kDa, a Stokes radius of 3.64 nm, and a slightly asymmetric shape with a frictional ratio f/fo = 1.43. As revealed during the purification steps and by immunoprecipitation experiments, the TeqR-like and TeqC-like proteins were not associated forming a heterooligomeric complex, differing from traditional PKA subunits. Co-immunoprecipitation results followed by mass spectrometry sequencing identified two isoforms of the parasite heat-shock protein 70, α-tubulin, and β-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum.

Regulation of Skn7-dependent, oxidative stress-induced genes by the RNA polymerase II-CTD phosphatase, Fcp1, and Mediator kinase subunit, Cdk8, in yeast

Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of fcp1 mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in fcp1 and cdk8Δ mutants and was required for the suppression of fcp1 mutant growth defects by loss of CDK8 under oxidative stress conditions. Targeted analysis revealed that mutating FCP1 decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes (TRX2 and TSA1) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of TRX2 and TSA1 observed in the fcp1 mutant. Interestingly, loss of CDK8 robustly normalized the mRNA levels of Skn7-dependent genes in the fcp1 mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of CDK8 could overcome transcriptional and/or posttranscriptional alterations in the fcp1 mutant through its regulatory effect on Skn7. Furthermore, our work also implicated FCP1 and CDK8 in the broader response to environmental stressors in yeast.

Cardiac ischemia-reperfusion injury induces ROS-dependent loss of PKA regulatory subunit RIα.

Type I PKA regulatory α-subunit (RIα; encoded by the Prkar1a gene) serves as the predominant inhibitor protein of the catalytic subunit of cAMP-dependent protein kinase (PKAc). However, recent evidence suggests that PKA signaling can be initiated by cAMP-independent events, especially within the context of cellular oxidative stress such as ischemia-reperfusion (I/R) injury. We determined whether RIα is actively involved in the regulation of PKA activity via reactive oxygen species (ROS)-dependent mechanisms during I/R stress in the heart. Induction of ex vivo global I/R injury in mouse hearts selectively downregulated RIα protein expression, whereas RII subunit expression appears to remain unaltered. Cardiac myocyte cell culture models were used to determine that oxidant stimulus (i.e., H2O2) alone is sufficient to induce RIα protein downregulation. Transient increase of RIα expression (via adenoviral overexpression) negatively affects cell survival and function upon oxidative stress as measured by increased induction of apoptosis and decreased mitochondrial respiration. Furthermore, analysis of mitochondrial subcellular fractions in heart tissue showed that PKA-associated proteins are enriched in subsarcolemmal mitochondria (SSM) fractions and that loss of RIα is most pronounced at SSM upon I/R injury. These data were supported via electron microscopy in A-kinase anchoring protein 1 (AKAP1)-knockout mice, where loss of AKAP1 expression leads to aberrant mitochondrial morphology manifested in SSM but not interfibrillar mitochondria. Thus, we conclude that modification of RIα via ROS-dependent mechanisms induced by I/R injury has the potential to sensitize PKA signaling in the cell without the direct use of the canonical cAMP-dependent activation pathway.NEW & NOTEWORTHY We uncovered a previously undescribed phenomenon involving oxidation-induced activation of PKA signaling in the progression of cardiac ischemia-reperfusion injury. Type I PKA regulatory subunit RIα, but not type II PKA regulatory subunits, is dynamically regulated by oxidative stress to trigger the activation of the catalytic subunit of PKA in cardiac myocytes. This effect may play a critical role in the regulation of subsarcolemmal mitochondria function upon the induction of ischemic injury in the heart.

Cyclin-Dependent Kinase Regulatory Subunit 2 Indicated Poor Prognosis and Facilitated Aggressive Phenotype of Hepatocellular Carcinoma.

Cyclin-dependent kinase regulatory subunit 2 (CKS2) is a member of the cell cycle-dependent protein kinase subunit family, which is implicated as an oncogene in various malignancies. However, the clinical significance, oncogenic functions, and related mechanisms of CKS2 in hepatocellular carcinoma (HCC) remain largely unclear. In the present study, expression features and prognostic value of CKS2 were evaluated in the bioinformatic databases and HCC tissues. The effects of CKS2 on the malignant phenotypes of HCC cells were explored in vitro. According to the analyses of three bioinformatic databases, mRNA levels of CKS2 were elevated in HCC tissues compared with the normal tissues. Immunohistochemical assays found that high CKS2 expression was closely associated with liver cirrhosis (P = 0.019), poor differentiation (P = 0.02), portal vein invasion (P < 0.001), TNM stage (P = 0.019), tumor metastasis (P = 0.008), and recurrence (P = 0.003). The multivariate regression analyses suggested that CKS2 was an independent prognostic factor for overall survival (HR = 2.088, P = 0.014) and disease-free survival (HR = 2.511, P = 0.002) of HCC patients. Moreover, the bioinformatic analyses indicated that CKS2 might be associated with the malignant phenotypes in HCC progression. In addition, in vitro assays showed that CKS2 expression was higher in HCC cell lines than in normal liver cells. Knockdown of CKS2 remarkably repressed the proliferation, colony formation (P = 0.0003), chemoresistance, migration (P = 0.0047), and invasion (P = 0.0012) of HCC cells. Taken together, overexpression of CKS2 was significantly correlated with poor prognosis of HCC patients and the malignant phenotypes of HCC cells, suggesting that it was a novel prognostic biomarker and potential target of HCC.

Intermittent hypoxia and hypercapnia induces inhibitor of nuclear factor-κB kinase subunit β-dependent atherosclerosis in pulmonary arteries.

Clinical studies have shown that obstructive sleep apnea (OSA) increases atherosclerosis risk. The inflammation, especially mediated by the macrophages via nuclear factor-κB (NF-κB), has been speculated to contribute to atherogenicity in OSA patients. Inhibitor of NF-κB kinase-β (IKKβ) is an essential element of the NF-κB pathway and is linked to atherosclerosis. We previously reported that atherosclerosis was accelerated in pulmonary artery (PA) but not in aorta when low-density lipoprotein receptor knockout (Ldlr-/-) mice were exposed to intermittent hypoxia/hypercapnia (IHH), a surrogate for recurrent upper-airway obstruction. Therefore, we hypothesized that IKKβ-dependent NF-κB activation in monocytes and macrophages plays a role in IHH-induced PA atherosclerosis. To test this hypothesis, myeloid restricted IKKβ deletion (IkkβΔMye) or control (IkkβF/F) mice were crossed with Ldlr-/- mice to generate double-knockout mice. Then, the mice were exposed to IHH or room air (Air) on high-fat diet for 8 or 16 wk. Lesions of PA and aorta were examined in IkkβΔMye;Ldlr-/- and IkkβF/F;Ldlr-/- male mice under IHH vs. Air. The results revealed that IKKβ deletion abolished IHH-induced PA atherosclerosis after 8-wk exposure but not after 16-wk exposure (8 wk: IkkβF/F;Ldlr-/-, IHH 13.5 ± 1.4 vs. Air 5.7 ± 0.7%, P < 0.01; IkkβΔMye;Ldlr-/-, IHH 7.4 ± 1.9% vs. Air 4.6 ± 1.3%, P = 0.24). Both IKKβ deletion and IHH had no effects on atherosclerosis in the aorta. Our findings demonstrate that IKKβ-dependent NF-κB activity in myeloid-lineage cells plays a critical role in IHH-induced PA atherosclerosis at the early stage.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation.

Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

CRISPR-Cas9-mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes.

Prkdc encodes for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) playing a key role in nonhomologous end joining pathway during DNA double-strand break repair and also influencing the homologous recombination (HR) repair system by phosphorylation of proteins involved in HR. In addition, Prkdc has other critical functions in biological processes, such as transcriptional regulation, telomere stability, apoptosis, and metabolism. DNA-PKcs upregulates during in vitro differentiation of mouse embryonic stem cells (mESCs). To address the potential role of Prkdc in mESCs pluripotency and in vitro differentiation into ectoderm, mesoderm, and endoderm germ layers under normal physiological conditions, a bi-allelic Prkdc-knockout cell line was generated in the present study by employing CRISPR/Cas9 system, and subsequently, its potential role in stemness and development was studied. The results of the study showed that the expression of pluripotency-associated genes, including Nanog and Sox-2 were overexpressed in the bi-allelic Prkdc-knockout cell line. Also, bi-allelic Prkdc-knockout cell line was shown to have typical mESCs cell morphology, cell cycle distribution, and alkaline phosphatase activity. Furthermore, the results of the study revealed that the expression of several germ layer markers is modulated in Prkdc-knockout lines. In conclusion, the findings of our study demonstrated the role of Prkdc during differentiation and development of ESCs.

Karyopherin MoKap119-mediated nuclear import of cyclin-dependent kinase regulator MoCks1 is essential for Magnaporthe oryzae pathogenicity.

Nuclear import of proteins relies on nuclear import receptors called importins/karyopherins (Kaps), whose functions were reported in yeasts, fungi, plants, and animal cells, including cell cycle control, morphogenesis, stress sensing/response, and also fungal pathogenecity. However, limited is known about the physiological function and regulatory mechanism of protein import in the rice-blast fungus Magnaporthe oryzae. Here, we identified an ortholog of β-importin in M. oryzae encoded by an ortholog of KAP119 gene. Functional characterisation of this gene via reverse genetics revealed that it is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The mokap119Δ mutant was also defective in formation of appressorium-like structure from hyphal tips. By affinity assay and liquid chromatography-tandem mass spectrometry, we identified potential MoKap119-interacting proteins and further verified that MoKap119 interacts with the cyclin-dependent kinase subunit MoCks1 and mediates its nuclear import. Transcriptional profiling indicated that MoKap119 may regulate transcription of infection-related genes via MoCks1 regulation of MoSom1. Overall, our findings provide a novel insight into the regulatory mechanism of M. oryzae pathogenesis likely by MoKap119-mediated nuclear import of the cyclin-dependent kinase subunit MoCks1.

Investigating the impact of local inflammation on granulosa cells and follicular development in women with ovarian endometriosis

To investigate the possible impact of local inflammation on granulosa cells (GCs) and follicular development in endometriosis patients. Prospective study with related paired design. Reproductive medicine center. A total of 80 endometriosis patients and 104 controls, with cultured GCs collected from control participants younger than 35years. Tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) inhibitor. Intrafollicular concentrations of cytokines measured with ELISA, NF-κB binding levels with electrophoretic mobility shift assay (EMSA), and telomerase activity (TA) with quantitative-telomeric repeat amplification protocol (Q-TRAP) assay, and protein and mRNA expression with Western blot and polymerase chain reaction analyses, respectively. Patients with endometriosis exhibited a statistically significantly lower antral follicle count (11.48 ± 8.11 vs. 15.68 ± 8.56), lower number of retrieved oocytes (8.28 ± 6.69 vs. 10.87 ± 6.26), and lower number of mature oocytes (6.67 ± 6.09 vs. 8.53 ± 5.69). The GCs from endometriosis patients showed higher NF-κB binding activity and increased expression of inhibitor of NF-κB kinase subunit β (IKKβ, 2.743-fold) and NF-κB inhibitor α (IκBα, 5.017-fold). Their NF-κB p65 expression was negatively associated with mature oocytes (bNF-κB' = -0.304, R2 = 0.195, R = 0.442) but positively associated with intrafollicular TNF-α (r = 0.37); TA showed a negative relationship with NF-κB binding levels (r = -0.667). Tumor necrosis factor-α induced expression of IκBα (5.408-fold) and NF-κB p65 (1.400-fold) but lowered human telomerase reverse transcriptase (hTERT) and TA levels (0.0009 vs. 0.5619) in cultured GCs. However, inhibiting NF-κB obviously increased hTERT expression (1.988-fold). Endometriosis showed activated NF-κB pathways in GCs, which might negatively affect TA and oocyte quality. Intrafollicular TNF-α might down-regulate TA and hTERT via NF-κB pathway, but further studies are required.

The role of PKAc1 in gene regulation and trichodimerol production in Trichoderma reesei

BackgroundTrichoderma reesei represents a model system for investigation of plant cell wall degradation and its connection to light response. The cyclic adenosine monophosphate pathway (cAMP pathway) plays an important role in both physiological outputs, being crucial for regulation of photoreceptor function as well as for cellulase regulation on different carbon sources. Phosphorylation of photoreceptors and of the carbon catabolite repressor CRE1 was shown in ascomycetes, indicating a relevance of protein kinase A in regulation of the target genes of these transcription factors as well as an impact on regulation of induction specific genes. Moreover, the cAMP pathway impacts growth and development.ResultsHere, we investigated gene regulation by the catalytic subunit of protein kinase A (PKAc1) upon growth on cellulose. We found distinct gene sets for regulation upon growth in light and darkness with an overlap of only 13 genes. PKAc1 regulates metabolic genes as well as transport and defense functions. The overlap of gene regulation by PKAc1 with the genes representing the cAMP dependent regulatory output of the photoreceptor ENV1 indicates an involvement of PKA in this pathway, which counteracts its effects by contrasting regulation. Moreover, we found considerable overlap with the gene sets regulated under cellulase inducing conditions and by the carbon catabolite repressor CRE1. Our analysis also showed that PKAc1 regulates the genes of the SOR cluster associated with the biosynthesis of sorbicillinoids. The homologue of gin4, encoding a CAMK type kinase, which is regulated by PKAc1, CRE1 and YPR2 showed a moderate impact on trichodimerol production. We isolated trichodimerol as representative sorbicillin compound and established a method for its quantification in large sample sets using high performance thin layer chromatography (HPTLC), which can be broadly applied for secondary metabolite screening of mutants or different growth conditions. Due to the high expression levels of the SOR cluster under conditions of sexual development we crosschecked the relevance of PKAc1 under these conditions. We could show that PKAc1 impacts biosynthesis of trichodimerol in axenic growth and upon mating.ConclusionsWe conclude that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.

Optimization study towards more potent thiazolidine-2,4-dione IKK-β modulator: Synthesis, biological evaluation and in silico docking simulation

Inhibition of IKK-β (inhibitor of nuclear factor kappa-B kinase subunit beta) has been broadly documentedas a promising approach for treatment of acute and chronic inflammatory diseases, cancer, and autoimmune diseases. Recently, we have identified a novel class of thiazolidine-2,4-diones as structurally novel modulators for IKK-β. Herein, we report a hit optimization study via analog synthesis strategy aiming to acquire more potent derivative(s), probe the structure activity relationship (SAR), and get reasonable explanations for the elicited IKK-β inhibitory activities though an in silico docking simulation study. Accordingly, a new series of eighteen thiazolidine-2,4-dione derivatives was rationally designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as noteworthy IKK-β potential modulators. Successfully, new IKK-β potent modulators were obtained, including the most potent analog up-to-date 7m with IC50 value of 260 nM. A detailed structure activity relationship (SAR) was discussed and a mechanistic study for 7m was carried out indicating its irreversible inhibition mode with IKK-β (Kinact value = 0.01 (min−1). Furthermore, the conducted in silico simulation study provided new insights for the binding modes of this novel class of modulators with IKK-β.