High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans.

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Target of rapamycin complex 2-dependent phosphorylation of the coat protein Pan1 by Akl1 controls endocytosis dynamics in Saccharomyces cerevisiae.

Target of rapamycin complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In the yeast Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. In this role, TORC2 regulates diverse processes, including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton, and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here, we report that Fpk1 mediates TORC2 signaling to control actin polarization, but not endocytosis, via aminophospholipid flippases. To search for specific targets of these flippase kinases, we exploited the fact that Fpk1 prefers to phosphorylate Ser residues within the sequence RXS(L/Y)(D/E), which is present ∼90 times in the yeast proteome. We observed that 25 of these sequences are phosphorylated by Fpk1 in vitro We focused on one sequence hit, the Ark/Prk-family kinase Akl1, as this kinase previously has been implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to preferentially inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which, in turn, reduces phosphorylation of Pan1 and of other endocytic coat proteins and ultimately contributes to a slowing of endocytosis kinetics. These results indicate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled through separate pathways that bifurcate at the level of the flippase kinases.

In vitro effects of FBXW7 mutation in serous endometrial cancer: Increased levels of potentially druggable proteins and sensitivity to SI-2 and dinaciclib.

Serous endometrial cancers (ECs) are clinically aggressive tumors that frequently harbor somatic mutations in FBXW7 (F-box and WD repeat domain-containing 7). The FBXW7 tumor suppressor is part of a SCF (complex of SKP1, Cullin 1, F-box protein) ubiquitin ligase complex which controls the degradation of numerous substrates that, if not properly regulated, can contribute to the initiation or progression of tumorigenesis. Despite reports that up to 30% of serous ECs include somatic mutations in FBXW7, the molecular effects of mutated FBXW7 in ECs have not been determined. Here, we used transient transfection and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing in serous EC cell lines to interrogate the molecular effects of six recurrent FBXW7 mutations. We show that FBXW7 mutations lead to increased Cyclin E1, steroid receptor coactivator 3 (SRC-3), c-MYC, Rictor, glycogen synthase kinase 3 (GSK3), P70S6 kinase, and protein kinase B (AKT) phosphorylated protein levels in serous EC cells. Furthermore, we demonstrate that CRISPR-edited FBXW7-mutant ARK1 serous EC cells exhibit increased sensitivity to SI-2 (a SRC inhibitor) and dinaciclib (a cyclin dependent kinase (CDK) inhibitor) compared to parental ARK1 cells. Collectively, our findings reveal biochemical effects of FBXW7 mutations in the context of EC and provide in vitro evidence of sensitivity to targeted inhibitors.

Partnership in action: The endoplasmic reticulum regulates the cytoskeleton.

The endoplasmic reticulum (ER) and the cytoskeleton are functionally linked in living cells. Past research has focused on how the cytoskeleton regulates ER dynamics. How the ER may contribute to cytoskeletal dynamics has been overlooked. In this commentary, I examined how the ER mediates actin and microtubule dynamics in plant cells.

Compatibility of Pupil Size Measured with Nidek ARK-1a Table Top Autorefractometer and Plusoptix A12C Photoscreener.

Aim: To evaluate the differences in pupil size measured with and without cycloplegia by a table top autorefractometer, ARK-1a Auto Ref/Keratometer, Nidek, Japan, 2014 release, compared to handheld photoscreener, Plusoptix A12C, software 6.1.12, under monocular and binocular conditions.Materials and Methods: In the study, 100 eyes of 100 patients ages 18-55 years were included. The patients' pupil sizes were measured with a table top autorefractometer, Nidek ARK 1a without cycloplegia, and then the pupils were remeasured under monocular and binocular conditions with a photoscreener, Plusoptix A12C, respectively. After that the measurements with cycloplegia were repeated in the same order. The values obtained with both devices were compared.Results: In the measurement results without cycloplegia, the pupil size measured by Nidek ARK 1a was found to be significantly different than that measured by Plusoptix A12C under both monocular and binocular conditions. Pupil size measured with Plusoptix A12C under monocular conditions was also different than pupil diameter measured under binocular conditions. In the measurements with cycloplegia, it was observed that the pupil size measured with Plusoptix A12C under monocular and binocular conditions was not different from the diameter measured with Nidek ARK 1a.Conclusion: Pupil sizes measured with Nidek ARK 1a and Plusoptix A12C in monocular and binocular conditions without cycloplegia are different, but measurements with cycloplegia are similar. We believe that pupil size measurement with Plusoptix A12C under binocular conditions without cycloplegia will be more appropriate when necessary as a screening device in clinical settings.

Enhancing the expression of ARK1 genes in poplar leads to multiple branches and transcriptomic changes.

The ARBORKNOX1 (ARK1) gene is an important gene for regulating plant growth and development; however, transcriptomic responses of enhancing expression of ARK1 gene in poplar are poorly investigated. To provide insight into the gene function of the ARK1 gene in poplar, the ARK1 transgenic poplar '717' and '84 K' lines were obtained, the morphology of transgenic plants was observed, and transcriptome profiles were compared. The results showed that there were multiple branches in ARK1 transgenic seedlings compared with non-transgenic seedlings. The results of transcriptome analysis showed that there were significant differences in transcriptome profiles between the transgenic lines of '717' and '84 K', and between non-transgenic lines (CK) and transgenic plants. The real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed the expression levels of the genes involved in the pathway of zeatin biosynthesis and brassinosteroid biosynthesis. The increase in expression levels of AHP and CYCD3 was related to multiple branches. Enhancing the expression of the ARK1 gene in poplar seedlings leads to multiple branches and transcriptomic changes.

Dual Inhibitory Action of a Novel AKR1C3 Inhibitor on Both Full-Length AR and the Variant AR-V7 in Enzalutamide Resistant Metastatic Castration Resistant Prostate Cancer.

The expanded use of second-generation antiandrogens revolutionized the treatment landscape of progressed prostate cancer. However, resistances to these novel drugs are already the next obstacle to be solved. Various previous studies depicted an involvement of the enzyme AKR1C3 in the process of castration resistance as well as in the resistance to 2nd generation antiandrogens like enzalutamide. In our study, we examined the potential of natural AKR1C3 inhibitors in various prostate cancer cell lines and a three-dimensional co-culture spheroid model consisting of cancer cells and cancer-associated fibroblasts (CAFs) mimicking enzalutamide resistant prostate cancer. One of our compounds, named MF-15, expressed strong antineoplastic effects especially in cell culture models with significant enzalutamide resistance. Furthermore, MF-15 exhibited a strong effect on androgen receptor (AR) signaling, including significant inhibition of AR activity, downregulation of androgen-regulated genes, lower prostate specific antigen (PSA) production, and decreased AR and AKR1C3 expression, indicating a bi-functional effect. Even more important, we demonstrated a persisting inhibition of AR activity in the presence of AR-V7 and further showed that MF-15 non-competitively binds within the DNA binding domain of the AR. The data suggest MF-15 as useful drug to overcome enzalutamide resistance.

Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation.

Extracellular matrix (ECM) remodeling, degradation and glioma cell motility are critical aspects of glioblastoma multiforme (GBM). Despite being a rich source of potential biomarkers and targets for therapeutic advance, the dynamic changes occurring within the extracellular environment that are specific to GBM motility have yet to be fully resolved. The gap junction protein connexin43 (Cx43) increases glioma migration and invasion in a variety of in vitro and in vivo models. In this study, the upregulation of Cx43 in C6 glioma cells induced morphological changes and the secretion of proteins associated with cell motility. Demonstrating the selective engagement of ECM remodeling networks, secretome analysis revealed the near-binary increase of osteopontin and matrix metalloproteinase-3 (MMP3), with gelatinase and NFF-3 assays confirming the proteolytic activities. Informatic analysis of interactome and secretome downstream of Cx43 identifies networks of glioma motility that appear to be synergistically engaged. The data presented here implicate ECM remodeling and matrikine signals downstream of Cx43/MMP3/osteopontin and ARK1B10 inhibition as possible avenues to inhibit GBM.

Inhibition of ß-ARK1 Ameliorates Morphine-induced Tolerance and Hyperalgesia Via Modulating the Activity of Spinal NMDA Receptors.

Our previous study has proposed that increased presynaptic NMDARs activities play pivotal roles in the development of opioid tolerance and hyperalgesia, and blocking spinal NMDARs attenuates chronic morphine-induced synaptic plasticity and behavior. However, the cellular signaling mechanisms remain to be investigated. The aim of this research was to address the role of ß-ARK1 in opioid analgesia. Opioid tolerance and hyperalgesia was induced by daily systemic morphine injections in rats for eight consecutive days. Whole-cell voltage-clamp was employed to record spontaneous EPSCs and evoked-AMPA-EPSCs in dorsal lamina II neurons. Strikingly, brief application of 1 µM morphine decreased the percentage of inhibition and was followed by a large LTP in the amplitude of monosynaptic evoked-AMPA-EPSCs in opioid-tolerant rats. There was no effect on these responses by postsynaptic dialysis of the G-protein inhibitor. Incubation with the NMDAR blocker AP5 potentiated morphine-induced inhibition and attenuated washout potentiation after cessation of morphine in the amplitude of AMPA-EPSCs. Incubation with ß-ARK1 inhibitor had the same effect on these responses. Incubation with ß-ARK1 inhibitor diminished NMDAR hyperfunction-increased glutamatergic synaptic transmission and enhanced the analgesic effect of morphine. Intrathecal injections of ß-ARK1 inhibitor significantly attenuated opioid-induced hyperalgesia and tolerance. ß-ARK1 plays a pivotal role in the development and maintenance of opioid tolerance and hyperalgesia. Blockade of ß-ARK1 activation ameliorates morphine tolerance and hyperalgesia via regulating the activity of spinal NMDARs. These findings provide electrophysiological evidence and useful insights regarding the mechanistic action of ß-ARK1 inhibitor as a potential anti-hyperalgesic agent to improve the efficacy of opioid therapies.

AXL modulates extracellular matrix protein expression and is essential for invasion and metastasis in endometrial cancer.

The receptor tyrosine kinase AXL promotes migration, invasion, and metastasis. Here, we evaluated the role of AXL in endometrial cancer. High immunohistochemical expression of AXL was found in 76% (63/83) of advanced-stage, and 77% (82/107) of high-grade specimens and correlated with worse survival in uterine serous cancer patients. In vitro, genetic silencing of AXL inhibited migration and invasion but had no effect on proliferation of ARK1 endometrial cancer cells. AXL-deficient cells showed significantly decreased expression of phospho-AKT as well as uPA, MMP-1, MMP-2, MMP-3, and MMP-9. In a xenograft model of human uterine serous carcinoma with AXL-deficient ARK1 cells, there was significantly less tumor burden than xenografts with control ARK1 cells. Together, these findings underscore the therapeutic potentials of AXL as a candidate target for treatment of metastatic endometrial cancer.

Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.