Substrate-binding loop interactions with pseudouridine trigger conformational changes that promote catalytic efficiency of pseudouridine kinase PUKI

Pseudouridine, one major RNA modification, is catabolized into uracil and ribose-5′-phosphate by two sequential enzymatic reactions. In the first step, pseudouridine kinase (PUKI) phosphorylates pseudouridine to pseudouridine 5′-monophosphate. High-fidelity catalysis of pseudouridine by PUKI prevents possible disturbance of in vivo pyrimidine homeostasis. However, the molecular basis of how PUKI selectively phosphorylates pseudouridine over uridine with >100-fold greater efficiency despite minor differences in their K_m values has not been elucidated. To investigate this selectivity, in this study we determined the structures of PUKI from Escherichia coli strain B (EcPUKI) in various ligation states. The structure of EcPUKI was determined to be similar to PUKI from Arabidopsis thaliana, including an α/β core domain and β-stranded small domain, with dimerization occurring via the β-stranded small domain. In a binary complex, we show that Ser30 in the substrate-binding loop of the small domain mediates interactions with the hallmark N1 atom of pseudouridine nucleobase, causing conformational changes in its quaternary structure. Kinetic and fluorescence spectroscopic analyses also showed that the Ser30-mediated interaction is a prerequisite for conformational changes and subsequent catalysis by EcPUKI. Furthermore, S30A mutation or EcPUKI complexed with other nucleosides homologous to pseudouridine but lacking the pseudouridine-specific N1 atom did not induce such conformational changes, demonstrating the catalytic significance of the proposed Ser30-mediated interaction. These analyses provide structural and functional evidence for a pseudouridine-dependent conformational change of EcPUKI and its functional linkage to catalysis.     

Dopamine depletion and subsequent treatment with L-DOPA, but not the long-lived dopamine agonist pergolide, enhances activity of the Akt pathway in the rat striatum

Dysregulation of signaling pathways is believed to contribute to Parkinson's disease pathology and l-DOPA-induced motor complications. Long-lived dopamine (DA) agonists are less likely to cause motor complications by virtue of continuous stimulation of DA receptors. In this study, we compared the effects of the unilateral 6-hydroxydopamine lesion and subsequent treatment with l-DOPA and DA agonist pergolide on signaling pathways in rats. Pergolide caused less pronounced behavioral sensitization than l-DOPA (25 mg/kg, i.p., 10 days), particularly at lower dose (0.5 and 0.25 mg/kg, i.p.). Pergolide, but not l-DOPA, reversed lesion-induced up-regulation of preproenkephalin and did not up-regulate preprodynorphine or DA D3 receptor in the lesioned hemisphere. Pergolide was as effective as l-DOPA in reversing the lesion-induced elevation of ERK2 phosphorylation in response to acute apomorphine administration (0.05 mg/kg, s.c.). Chronic l-DOPA significantly elevated the level of Akt phosphorylation at both Thr(308) and Ser(473) and concentration of phosphorylated GSK3alpha, whereas pergolide suppressed the lesion- and/or challenge-induced supersensitive Akt responses. The data indicate that l-DOPA, unlike pergolide, exacerbates imbalances in the Akt pathway caused by the loss of DA. The results support the hypothesis that the Akt pathway is involved in long-term actions of l-DOPA and may be linked to l-DOPA-induced dyskinesia.     

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24 h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.     

Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior

Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCɛ) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCɛ activator, ψɛRACK, increased levels of pro-CRF, whereas reducing PKCɛ levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCɛ by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCɛ^−/− mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCɛ control over anxiety-like behavior through regulation of CRF in the amygdala.     

An engram found? Evaluating the evidence from fruit flies

Is it possible to localize a memory trace to a subset of cells in the brain? If so, it should be possible to show: first, that neuronal plasticity occurs in these cells. Second, that neuronal plasticity in these cells is sufficient for memory. Third, that neuronal plasticity in these cells is necessary for memory. Fourth, that memory is abolished if these cells cannot provide output during testing. And fifth, that memory is abolished if these cells cannot receive input during training. With regard to olfactory learning in flies, we argue that the notion of the olfactory memory trace being localized to the Kenyon cells of the mushroom bodies is a reasonable working hypothesis.     

Rice Crinkly4 receptor-like kinase positively regulates culm elongation and amino acid K532 is not essential for its kinase activity

Receptor-like kinases (RLKs) play important roles in multiple aspects of plant growth and development. As a member of the TNFR-like RLK subfamily, rice Crinkly4 (OsCR4) functions mainly in epidermal cell differentiation in many organs. Here we show that in addition to its essential role in epidermal cell differentiation in the palea and lemma, OsCR4 positively regulates rice culm elongation, similar to maize CR4. Although OsCR4 is an active kinase, like CR4 in maize and ACR4 in Arabidopsis, the conserved amino acid K532 in OsCR4 is not essential for its kinase activity in vitro. Whether other conserved amino acids are required for its kinase activity and the relationship between its activity and function in plant development remain to be investigated.     

Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer

Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P = 0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P = 0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G₂/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback loop. Further, inhibition of polo-like kinase 1 could enhance the sensitivity of non-small cell lung cancer cells to taxanes or irradiation. Thus, polo-like kinase 1 might be a prognostic marker and a chemo- or radiotherapeutic target for non-small cell lung cancer.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.