scholarly journals Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

2017 ◽  
Author(s):  
Rafael M. Couñago ◽  
Charles K. Allerston ◽  
Pavel Savitsky ◽  
Hatylas Azevedo ◽  
Paulo H. Godoi ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Related Protein ◽  
Chemical Probes ◽  
Cellular Processes ◽  
Starting Point ◽  
Loop Conformations ◽  
Potential Use ◽  
Specific Inhibitors

ABSTRACTThe human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

scholarly journals Mass Spectrometry-Based Proteomic Characterization of Cutaneous Melanoma Ectosomes Reveals the Presence of Cancer-Related Molecules

2020 ◽  
Vol 21 (8) ◽  
pp. 2934 ◽  
Author(s):  
Magdalena Surman ◽  
Sylwia Kędracka-Krok ◽  
Dorota Hoja-Łukowicz ◽  
Urszula Jankowska ◽  
Anna Drożdż ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Content ◽  
Cell Lines ◽  
Cutaneous Melanoma ◽  
Epithelial Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Starting Point ◽  
Novel Biomarkers

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC–MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial–mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

Differential targeting of cPKC and nPKC decodes and regulates Ca2+ and lipid signalling

2014 ◽  
Vol 42 (6) ◽  
pp. 1538-1542 ◽  
Author(s):  
Xin Hui ◽  
Lars Kaestner ◽  
Peter Lipp
Keyword(s):  
Protein Kinases ◽  
Consensus Sequence ◽  
Membrane Targeting ◽  
Cellular Processes ◽  
Lipid Signalling ◽  
Pkc Isoforms

Protein kinases C (PKCs) are ubiquitously expressed and play critical roles in a plethora of physiological and pathophysiological processes. Owing to PKCs’ highly conserved phosphorylation consensus sequence, it has been difficult to distinguish the role of individual PKC isoforms. Recently, the identification of novel membrane targeting via subcellularly targeted diacylglycerol production found for novel PKCs (nPKCs), together with a characterization of their putative functions, has shed new light on the specific roles of individual PKCs in cellular processes.

scholarly journals Isolation and Characterization of Broad Spectrum Coaggregating Bacteria from Different Water Systems for Potential Use in Bioaugmentation

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e94220 ◽  
Author(s):  
Zhongqin Cheng ◽  
Xiangxun Meng ◽  
Haichao Wang ◽  
Mei Chen ◽  
Mengying Li
Keyword(s):  
Broad Spectrum ◽  
Water Systems ◽  
Isolation And Characterization ◽  
Potential Use

scholarly journals Lipid Oxidation in Emulsions Fortified with Iron-Loaded Alginate Beads

Foods ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 361 ◽  
Author(s):  
Alime Cengiz ◽  
Karin Schroën ◽  
Claire Berton-Carabin
Keyword(s):  
Lipid Oxidation ◽  
Ferrous Sulfate ◽  
Continuous Phase ◽  
Alginate Beads ◽  
Reactive Iron ◽  
Fortified Food ◽  
Starting Point ◽  
Oil In Water ◽  
Potential Use

The potential use of iron-loaded alginate beads to fortify oil-in-water (O/W) emulsions was studied. Iron-loaded alginate beads with different sizes (0.65, 0.84, 1.5 and 2 mm) were produced by ionic gelation with calcium chloride, leading to 81% encapsulation efficiency (EE) of ferrous sulfate. These beads were added to O/W emulsions to investigate their effect on lipid oxidation. The use of iron-loaded alginate beads inhibited lipid oxidation in emulsions, compared to a control emulsion with the same concentration of free ferrous sulfate in the continuous phase, but did not totally prevent it. Results obtained with scanning electron microscopy and energy dispersive X-ray spectroscopy (EDX) analysis showed that some reactive iron was present at the surface of the beads. Oxidation of the lipid droplets was slightly higher for smaller alginate beads, suggesting that the reaction could be linked to the total bead surface. When covering iron-loaded beads with an extra layer of alginate, lipid oxidation was inhibited, which confirmed the role of reactive surface-bound iron. This study shows that the location of iron within the encapsulates plays a crucial role in the chemical stability of fortified foods and should be taken as a starting point in the design of iron-fortified food products.

scholarly journals The specifi cs of Monotheism (tawhid) as “a thing-in-itself”. On the Evolution of Islamic Theology in the Late Russian Empire

2022 ◽  
Vol 14 (4) ◽  
pp. 866-882
Author(s):  
D. R. Gilmutdinov
Keyword(s):  
Russian Empire ◽  
Religious Epistemology ◽  
Dynamic Characterization ◽  
Islamic Theology ◽  
Xviii Century ◽  
Starting Point ◽  
Thing In Itself ◽  
The Subject

In this paper, we will try to give a dynamic characterization of the object and the subject of Modern theology among the Tatar Muslims on the exemplar of the theological views of ‘Abdunnasīr Qursavi (1776–1812), Shihabutdin Marjani (1818–1889) and Murad Ramzi (1854–1934) (and partly of their contemporaries). The incognizability of the Creator and the faith as “a thing-in-itself” transformed Tatar Religious Epistemology into the cognition of more defi nite realities. Agnosticism in the question of God’s attributes led to the anthropocentric features of theological worldviews. The above-mentioned chain of theologians demonstrates not only the continuity of the Tatar Theology, but also refl ects the dynamics of the evolution of the attitude towards the madhhabs and towards the role of an individual, the specifi cs of the Naqshbandi-Mujaddidiya Sufi brotherhood, as well as the Ottoman ‘usul fi qh’ in the modernization period of the early XVIII century. In general, the works of Qursavi constitute a certain system of views that can be considered as a certain cornerstone, the so-called ‘starting point’ of Tatar School of Theology.

scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

2019 ◽  
Author(s):  
Timothy S. Strutzenberg ◽  
Ruben Garcia-Ordonez ◽  
Scott Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Orphan Nuclear Receptor ◽  
Chemical Probes ◽  
Structural Determinants ◽  
Synthetic Ligand ◽  
Hdx Ms

ABSTRACTMembers of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism1 to inflammation2 and cancer3. As ligand-gated transcription factors, synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology4. However, nearly half of NRs do not have specific cognate ligands or its unclear if they possess ligand dependent activities and these receptors are called orphans. Here we demonstrate that ligand-dependent action of the orphan nuclear receptor RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings are corroborated with X-ray co-crystallography and site-directed mutagenesis to collectively reveal the structural determinants of RORγ ligand-dependent activation, critical for designing full agonists for application in cancer immunotherapy. Combined these observations support a model of receptor activation to more accurately describe RORγ pharmacology. Likewise, this ‘bump-and-hole’ inspired approach could be extended to other orphan NRs to explore the ligand-dependent activities that are important for defining pharmacology.

scholarly journals ERKs - a New Marker in Psychiatry?

2009 ◽  
Vol 24 (S1) ◽  
pp. 1-1
Author(s):  
M. Mateus ◽  
G.P. da Silva Borges ◽  
C. Silva ◽  
F. Lourenço
Keyword(s):  
Animal Models ◽  
Current Literature ◽  
Biological Markers ◽  
Antidepressant Effect ◽  
Clinical Use ◽  
Biological Mechanisms ◽  
Cellular Processes ◽  
Extracellular Signal ◽  
Specific Inhibitors

Recent studies involved the pathways of kinases regulated by extracellular signal (ERK - extracellular signal regulated kinases), a broad range of key cellular processes, in the mechanisms of depression and consequently in the action of antidepressants. It is also known that the use of specific inhibitors of phosphorylation of ERKs1 / 2 showed to have antidepressant effect in animal models. Fluoxetina (SSRI) was recently discovered to be a potente inhibitor of phosphorylation of ERKs. The ERKs1 / 2 and recently the 3, are present in neurons and glia, these also engaged in biological mechanisms of depression.The authors propose to do, based on the current literature, the characterization of the type (s) of cell (s) where changes in activation of ERKs1 / 2, occur during depression, and during the administration of antidepressants, in order to understand, to what extent these kinases may be considered as biological markers of depression. Possibly also to examine the feasibility of using these markers in clinical use.

scholarly journals Bent DNA Bows as Sensing Amplifiers for Detecting DNA-Interacting Salts and Molecules

Sensors ◽  
2020 ◽  
Vol 20 (11) ◽  
pp. 3112
Author(s):  
Jack Freeland ◽  
Lihua Zhang ◽  
Shih-Ting Wang ◽  
Mason Ruiz ◽  
Yong Wang
Keyword(s):  
Organic Molecules ◽  
Inorganic Salts ◽  
Dna Interactions ◽  
Bent Dna ◽  
Small Organic Molecules ◽  
Cellular Processes ◽  
Economic Methods ◽  
Simple Economic ◽  
Potential Use

Due to the central role of DNA, its interactions with inorganic salts and small organic molecules are important. For example, such interactions play important roles in various fundamental cellular processes in living systems and are involved in many DNA-damage related diseases. Strategies to improve the sensitivity of existing techniques for studying DNA interactions with other molecules would be appreciated in situations where the interactions are too weak. Here we report our development and demonstration of bent DNA bows for amplifying, sensing, and detecting the interactions of 14 inorganic salts and small organic molecules with DNA. With the bent DNA bows, these interactions were easily visualized and quantified in gel electrophoresis, which were difficult to measure without bending. In addition, the strength of the interactions of DNA with the various salts/molecules were quantified using the modified Hill equation. This work highlights the amplification effects of the bending elastic energy stored in the DNA bows and the potential use of the DNA bows for quantitatively measuring DNA interactions with small molecules as simple economic methods; it may also pave the way for exploiting the bent DNA bows for other applications such as screening DNA-interacting molecules and drugs.

NH2 terminus of serum and glucocorticoid-regulated kinase 1 binds to phosphoinositides and is essential for isoform-specific physiological functions

2007 ◽  
Vol 292 (6) ◽  
pp. F1741-F1750 ◽  
Author(s):  
Alan C. Pao ◽  
James A. McCormick ◽  
Hongyan Li ◽  
John Siu ◽  
Cedric Govaerts ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Kinase Activity ◽  
Regulatory Protein ◽  
Forkhead Transcription Factor ◽  
Cellular Processes ◽  
Cellular Compartments ◽  
Stimulation Of

Serum and glucocorticoid regulated kinase 1 (SGK1) has been identified as a key regulatory protein that controls a diverse set of cellular processes including sodium (Na+) homeostasis, osmoregulation, cell survival, and cell proliferation. Two other SGK isoforms, SGK2 and SGK3, have been identified, which differ most markedly from SGK1 in their NH2-terminal domains. We found that SGK1 and SGK3 are potent stimulators of epithelial Na+ channel (ENaC)-dependent Na+ transport, while SGK2, which has a short NH2 terminus, is a weak stimulator of ENaC. Further characterization of the role of the SGK1 NH2 terminus revealed that its deletion does not affect in vitro kinase activity but profoundly limits the ability of SGK1 either to stimulate ENaC-dependent Na+ transport or inhibit Forkhead-dependent gene transcription. The NH2 terminus of SGK1, which shares sequence homology with the phosphoinositide 3-phosphate [PI( 3 )P] binding domain of SGK3, binds phosphoinositides in protein lipid overlay assays, interacting specifically with PI( 3 )P, PI( 4 )P, and PI( 5 )P, but not with PI( 3 , 4 , 5 )P3. Moreover, a point mutation that reduces phosphoinositide binding to the NH2 terminus also reduces SGK1 effects on Na+ transport and Forkhead activity. These data suggest that the NH2 terminus, although not required for PI 3-kinase-dependent modulation of SGK1 catalytic activity, is required for multiple SGK1 functions, including stimulation of ENaC and inhibition of the proapoptotic Forkhead transcription factor. Together, these observations support the idea that the NH2-terminal domain acts downstream of PI 3-kinase-dependent activation to target the kinase to specific cellular compartments and/or substrates, possibly through its interactions with a subset of phosphoinositides.

scholarly journals Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus

2021 ◽  
Vol 22 (18) ◽  
pp. 9945
Author(s):  
Luisa Galla ◽  
Nicola Vajente ◽  
Diana Pendin ◽  
Paola Pizzo ◽  
Tullio Pozzan ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Processes ◽  
A Cell ◽  
Dynamic Concentration ◽  
Subcellular Compartmentalization

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

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