scholarly journals The novel ORFV protein ORFV113 activates LPA-p38 signaling

2021 ◽  
Vol 17 (10) ◽  
pp. e1009971
Author(s):  
Sushil Khatiwada ◽  
Gustavo Delhon ◽  
Sabal Chaulagain ◽  
Daniel L. Rock
Keyword(s):  
Kinase Inhibitor ◽  
Cellular Functions ◽  
P38 Kinase ◽  
Wide Range ◽  
Viral Plaque ◽  
Infected Cells ◽  
Kinase Phosphorylation ◽  
In Cells

Viruses have evolved mechanisms to subvert critical cellular signaling pathways that regulate a wide range of cellular functions, including cell differentiation, proliferation and chemotaxis, and innate immune responses. Here, we describe a novel ORFV protein, ORFV113, that interacts with the G protein-coupled receptor Lysophosphatidic acid receptor 1 (LPA1). Consistent with its interaction with LPA1, ORFV113 enhances p38 kinase phosphorylation in ORFV infected cells in vitro and in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. Infection of cells with virus lacking ORFV113 (OV-IA82Δ113) significantly decreased p38 phosphorylation and viral plaque size. Infection of cells with ORFV in the presence of a p38 kinase inhibitor markedly diminished ORFV replication, highlighting importance of p38 signaling during ORFV infection. ORFV113 enhancement of p38 activation was prevented in cells in which LPA1 expression was knocked down and in cells treated with LPA1 inhibitor. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host. Notably, ORFV113 represents the first viral protein that modulates p38 signaling via interaction with LPA1 receptor.

A Review on Application of Novel Solid Nanostructures in Drug Delivery

2018 ◽  
Vol 53 ◽  
pp. 22-36 ◽  
Author(s):  
Habibollah Faraji ◽  
Reza Nedaeinia ◽  
Esmaeil Nourmohammadi ◽  
Bizan Malaekeh-Nikouei ◽  
Hamid Reza Sadeghnia ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Drug Therapy ◽  
Biomedical Applications ◽  
Imaging Biomarkers ◽  
Cellular Functions ◽  
Scientific Innovation ◽  
Wide Range ◽  
Targeted Drug

Nanotechnology as a multidisciplinary and scientific innovation plays an important role in numerous biomedical applications, such as molecular imaging, biomarkers and biosensors and also drug delivery. A wide range of studies have been conducted on using of nanoparticles for early diagnosis and targeted drug therapy of various diseases. In fact, the small size, customized surface, upgraded solubility, or multi-functionality of nanoparticles enabled them to interact with complex cellular functions in new ways which opened many doors and created new biomedical applications. These studies demonstrated that nanotechnology vehicles can formulate biological products effectively, and this nano-formulated products with a potent ability against different diseases, were represented to have better biocompatibility, bioaccessibility and efficacy, under in vitro and in vivo conditions.

scholarly journals Negative Regulation of ASK1 by p21Cip1 Involves a Small Domain That Includes Serine 98 That Is Phosphorylated by ASK1 In Vivo

2007 ◽  
Vol 27 (9) ◽  
pp. 3530-3541 ◽  
Author(s):  
Jun Zhan ◽  
John B. Easton ◽  
Shile Huang ◽  
Ashutosh Mishra ◽  
Limin Xiao ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cellular Functions ◽  
Terminal Protein ◽  
Jnk Pathway ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Small Domain ◽  
Cellular Stresses

ABSTRACT The cyclin-dependent kinase inhibitor p21Cip1 regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21Cip1. Here we show that small deletions of p21Cip1 around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21Cip1 and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21Cip1 is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21Cip1, predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21Cip1 to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98.

scholarly journals Strong In Vivo Inhibition of HIV-1 Replication by Nullbasic, a Tat Mutant

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Hongping Jin ◽  
Yifan Sun ◽  
Dongsheng Li ◽  
Min-Hsuan Lin ◽  
Mary Lor ◽  
...  
Keyword(s):  
Mutant Form ◽  
Mrna Levels ◽  
Functional Cure ◽  
Fold Reduction ◽  
Infected Cells ◽  
Hiv 1 ◽  
Transcription And Replication ◽  
In Cells

ABSTRACT Nullbasic is a mutant form of the HIV-1 transcriptional activator protein (Tat) that strongly inhibits HIV-1 transcription and replication in lymphocytes in vitro. To investigate Nullbasic inhibition in vivo, we employed an NSG mouse model where animals were engrafted with primary human CD4+ cells expressing a Nullbasic-ZsGreen1 (NB-ZSG) fusion protein or ZSG. NB-ZSG and ZSG were delivered by using a retroviral vector where CD4+ cells were transduced either prior to (preinfection) or following (postinfection) HIV-1 infection. The transduced cells were analyzed in vitro up to 10 days postinfection (dpi) and in vivo up to 39 dpi. Compared to ZSG, NB-ZSG strongly inhibited HIV-1 replication both in vitro and in vivo using preinfection treatment. In vitro, HIV-1 mRNA levels in cells were reduced by up to 60-fold. In vivo, HIV-1 RNA was undetectable in plasma samples during the course of the experiment, and HIV-1 mRNA levels in resident CD4+ cells in organ tissue were reduced up to 2,800-fold. Postinfection treatment of HIV-1-infected cells with NB-ZSG attenuated HIV-1 infection for up to 14 days. In vitro, a 25-fold reduction of viral mRNA in cells was observed but diminished to a <2-fold reduction by 10 dpi. In vivo, HIV-1 RNA was undetectable in plasma of NB-ZSG mice at 14 dpi but afterwards was not significantly different between NB-ZSG mice and control mice. However, we observed higher levels of CD4+ cells in NB-ZSG mice than in control mice, suggesting that NB-ZSG imparted a survival advantage to HIV-1-infected animals. IMPORTANCE HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces viral loads below detectable levels in patients. However, therapy interruption leads to viral rebound due to latently infected cells, which serve as a source of continued viral infection. Interest in strategies leading to a functional cure for HIV-1 infection by long-term or permanent viral suppression is growing. Here, we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, inhibits HIV-1 transcription in infected CD4+ cells in vivo. Analysis shows that stable expression of Nullbasic in CD4+ cells could lead to durable anti-HIV-1 activity. Nullbasic, as a gene therapy candidate, could be a part of a functional-cure strategy to suppress HIV-1 transcription and replication.

scholarly journals Inhibition of SUMO-independent PML oligomerization by the human cytomegalovirus IE1 protein

2006 ◽  
Vol 87 (8) ◽  
pp. 2181-2190 ◽  
Author(s):  
Heejung Kang ◽  
Eui Tae Kim ◽  
Hye-Ra Lee ◽  
Jung-Jin Park ◽  
Yoon Young Go ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Insect Cells ◽  
Inhibitory Effect ◽  
Sumo Protease ◽  
Sumo Proteases ◽  
Specific Protease ◽  
Infected Cells ◽  
In Cells

In human cytomegalovirus-infected cells, the immediate-early IE1 protein disrupts the subnuclear structures known as the PML oncogenic domains or PODs, via the induction of PML desumoylation. This activity correlates with the functions of IE1 in transcriptional regulation and in the stimulation of lytic infection. Here, the effects of IE1 in induction of desumoylation of PML were characterized. IE1 did not interfere with the formation of sumoylated forms of PML in vitro. In in vitro assays using the sumoylated proteins, a SUMO-specific protease SENP1 desumoylated both PML and IE1. However, the IE1 proteins generated from bacteria or insect cells were unable to desumoylate PML in the same conditions. Although both IE1 and SUMO proteases such as SENP1, Axam and SuPr-1 efficiently desumoylated PML in co-transfection assays, they exerted different effects on the localization of PML. In cells transfected with either SENP1 or SuPr-1, the number of PML foci was reduced significantly and these remnant PML foci were devoid of SUMO-1 signals. However, in cells co-transfected with both SUMO proteases and IE1, these SUMO-independent PML foci were also completely disrupted. Furthermore, IE1, but not SENP1, was shown to disrupt the PML foci generated via transfection of a sumoylation-deficient mutant of PML. These data suggest that IE1 exhibits neither an inhibitory effect on sumoylation of PML nor intrinsic SUMO protease activity against PML in vitro. The finding that IE1 is capable of disrupting SUMO-independent PML aggregates suggests that inhibition of PML oligomerization by IE1 may play an important role in inducing PML desumoylation in vivo.

scholarly journals NcGRA2as a molecular target to assess the parasiticidal activity of toltrazuril againstNeospora caninum

Parasitology ◽  
2008 ◽  
Vol 135 (9) ◽  
pp. 1065-1073 ◽  
Author(s):  
M. STROHBUSCH ◽  
N. MÜLLER ◽  
A. HEMPHILL ◽  
G. GREIF ◽  
B. GOTTSTEIN
Keyword(s):  
Real Time ◽  
Neospora Caninum ◽  
Day Treatment ◽  
Mrna Level ◽  
Live Cells ◽  
Wide Range ◽  
Biological Criteria ◽  
Infected Cells

SUMMARYThe treatment ofNeospora caninuminfection in the bovine host is still at an experimental stage. In contrast to thein vivosituation, a wide range of compounds have been intensively investigated in cell-culture-based assays. Tools to demonstrate efficacy of treatment have remained conventional including morphological and cell biological criteria. In this work, we present a molecular assay that allows the distinction between live and dead parasites. Live parasites can be detected by measuring the mRNA level of specific genes, making use of the specific mRNA available in live cells. TheNcGra2gene ofN. caninum, which is known to be expressed in both tachyzoites and bradyzoites, was used to establish a quantitative real-time RT-PCR, for monitoring parasite viability. Validation of the systemin vitrowas achieved usingNeospora-infected cells that had been treated for 2–20 days with 30 μg/ml toltrazuril.NcGRA2-RT-real time PCR demonstrated that a 10-day toltrazuril-treatment exerted parasitostatic activity, as assessed by the presence ofNcGRA2-transcripts, whereas after a 14-day treatment period noNcGRA2-transcripts were detected, showing that the parasites were no longer viable. Concurrently, extended culture for a period of 4 weeks in the absence of the drug following the 14-day toltrazuril treatment did not lead to further parasite proliferation, confirming the parasiticidal effect of the treatment. This assay has the potential to be widely used in the development of novel drugs againstN. caninum, with a view to distinguishing between parasiticidal and parasitostatic efficacy of given compounds.

scholarly journals Signaling roles of phosphoinositides in the retina

2020 ◽  
pp. jlr.TR120000806 ◽  
Author(s):  
Raju V. S. Rajala
Keyword(s):  
Cell Types ◽  
Cellular Functions ◽  
Parent Molecule ◽  
Phosphoinositide Signaling ◽  
Wide Range ◽  
The Many ◽  
And Function ◽  
Different Cell Types

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (PIs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of PI kinases and PI phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule phosphatidylinositol. PI signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane binding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIs in general, in this review, we discuss recent studies and advances in PI lipid signaling in the retina. We specifically focus on PI lipids from vertebrate (e.g. bovine, rat, mice, toad, and zebrafish) and invertebrate (e.g. drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIs revealed from animal models and human diseases, and methods to study PI levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PI-modifying enzymes/phosphatases and further unravel PI regulation and function in the different cell types of the retina.

scholarly journals Characterization of the Rhesus Cytomegalovirus US28 Locus

2003 ◽  
Vol 77 (19) ◽  
pp. 10404-10413 ◽  
Author(s):  
M. E. T. Penfold ◽  
T. L. Schmidt ◽  
D. J. Dairaghi ◽  
P. A. Barry ◽  
T. J. Schall
Keyword(s):  
Viral Entry ◽  
Intracellular Signaling ◽  
Northern Analysis ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Wide Range ◽  
Infected Cells ◽  
G Protein Coupled

ABSTRACT Human cytomegalovirus (CMV) US28 (and the related open reading frame [ORF] US27) are G-protein-coupled receptor homologs believed to play a role in viral pathogenesis. In vitro, US28 has been shown to bind and internalize ligands, as well as activate intracellular signaling in response to certain chemokines, and to initiate the migration of smooth muscle cells to chemokine gradients. To assess the role of US28 in vivo, we examined the rhesus model and sequenced and characterized the rhesus CMV US28 locus. We found that rhesus CMV carries five tandem homologs of US28, all widely divergent from US28 and from each other. By reverse transcription-PCR and Northern analysis, we demonstrated expression of these ORFs in infected cells. With stable cell lines expressing these ORFs, we analyzed the homolog's binding and signaling characteristics across a wide range of chemokines and found one (RhUS28.5) to have a ligand binding profile similar to that of US28. In addition, we localized US28 and the rhesus CMV homolog RhUS28.5 to the envelope of infectious virions, suggesting a role in viral entry or cell tropism.

scholarly journals The SUMO-specific isopeptidase SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic α-helix

2018 ◽  
Vol 29 (15) ◽  
pp. 1878-1890 ◽  
Author(s):  
Hana M. Odeh ◽  
Etienne Coyaud ◽  
Brian Raught ◽  
Michael J. Matunis
Keyword(s):  
Binding Assays ◽  
Cellular Functions ◽  
Intracellular Membranes ◽  
Vitro Binding ◽  
Wide Range ◽  
Associated Functions ◽  
Associated Proteins ◽  
Α Helix

Sumoylation regulates a wide range of essential cellular functions, many of which are associated with activities in the nucleus. Although there is also emerging evidence for the involvement of the small ubiquitin-related modifier (SUMO) at intracellular membranes, the mechanisms by which sumoylation is regulated at membranes is largely unexplored. In this study, we report that the SUMO-specific isopeptidase, SENP2, uniquely associates with intracellular membranes. Using in vivo analyses and in vitro binding assays, we show that SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic α-helix that promotes direct membrane binding. Furthermore, we demonstrate that SENP2 binding to intracellular membranes is regulated by interactions with the nuclear import receptor karyopherin-α. Consistent with membrane association, biotin identification (BioID) revealed interactions between SENP2 and endoplasmic reticulum, Golgi, and inner nuclear membrane-associated proteins. Collectively, our findings indicate that SENP2 binds to intracellular membranes where it interacts with membrane-associated proteins and has the potential to regulate their sumoylation and membrane-associated functions.

scholarly journals MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

2012 ◽  
Vol 287 (42) ◽  
pp. 35127-35138 ◽  
Author(s):  
Christian Delphin ◽  
Denis Bouvier ◽  
Maxime Seggio ◽  
Emilie Couriol ◽  
Yasmina Saoudi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Temperature Sensor ◽  
Cold Sensitivity ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Microtubule Network ◽  
Wide Range ◽  
In Cells

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

scholarly journals Specific inhibition of skeletal α-actin gene transcription by applied mechanical forces through integrins and actin

1999 ◽  
Vol 341 (3) ◽  
pp. 647-653 ◽  
Author(s):  
April M. LEW ◽  
Michael GLOGAUER ◽  
Christopher A. G. MCCULLOCH
Keyword(s):  
Actin Cytoskeleton ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Applied Force ◽  
Mechanical Forces ◽  
Transcriptional Enhancer ◽  
P38 Kinase ◽  
Applied Forces ◽  
In Cells

Skeletal α-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5′-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/μm2) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca2+-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by force; indeed, the p38 kinase inhibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an intact actin cytoskeleton and can be mapped to two different response elements. This inhibition might be mediated through the p38 kinase.

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