scholarly journals Interaction between PHB2 and Enterovirus A71 VP1 Induces Autophagy and Affects EV-A71 Infection

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 414 ◽  
Author(s):  
Weitao Su ◽  
Shan Huang ◽  
Huimin Zhu ◽  
Bao Zhang ◽  
Xianbo Wu
Keyword(s):  
Defense Mechanism ◽  
Foot And Mouth Disease ◽  
Underlying Mechanism ◽  
Host Protein ◽  
Mass Spectrometry Analysis ◽  
Structural Protein ◽  
Viral Protein Synthesis ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Enterovirus A71

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD). HFMD caused by EV-A71 seriously endangers children’s health. Although autophagy is an important antiviral defense mechanism, some viruses have evolved strategies to utilize autophagy to promote self-replication. EV-A71 can utilize autophagy vesicles as replication scaffolds, indicating that EV-A71 infection is closely related to its autophagy induction mechanism. VP1, a structural protein of EV-A71, has been reported to induce autophagy, but the underlying mechanism is still unclear. In this study, we found that the C-terminus (aa 251–297) of VP1 induces autophagy. Mass spectrometry analysis suggested that prohibitin 2 (PHB2) interacts with the C-terminus of the EV-A71 VP1 protein, and this was further verified by coimmunoprecipitation assays. After PHB2 knockdown, EV-A71 replication, viral particle release, and viral protein synthesis were reduced, and autophagy was inhibited. The results suggest that PHB2 interaction with VP1 is essential for induction of autophagy and the infectivity of EV-A71. Furthermore, we confirmed that EV-A71 induced complete autophagy that required autolysosomal acidification, thus affecting EV-A71 infection. In summary, this study revealed that the host protein PHB2 is involved in an autophagy mechanism during EV-A71 infection.

scholarly journals Foot-and-mouth disease virus non-structural protein 3A modulates cellular innate immune responses to influence host tropism

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Soumendu Chakravarti ◽  
Caroline Wright ◽  
Emma Howes ◽  
Richard Kock ◽  
Terry Jackson ◽  
...  
Keyword(s):  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Economic Losses ◽  
Structural Protein ◽  
Host Tropism ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Species Specific

The picornavirus foot-and-mouth disease virus (FMDV) is responsible for one of the most significant diseases of livestock, leading to large economic losses due to reduced productivity and trade embargoes for areas not certified as disease-free. The picornavirus non-structural protein 3A is involved in replication of the viral RNA genome and is implicated in host tropism of several picornaviruses. Deletions in the C-terminus of 3A have been observed in FMDV outbreaks specific for swine and such viruses are non-pathogenic in cattle. The mechanism for species specific attenuation of FMDV is unknown. We have shown that FMDV containing a C-terminal deletion in 3A is attenuated in bovine cell culture and that the attenuated phenotype can be reversed by the JAK1/2 inhibitor Ruxolitinib (Rux), identifying a role for the induction of interferon stimulated genes (ISGs) in the restricted bovine tropism of the 3A-deleted virus.

scholarly journals Functional Insights into Silymarin as an Antiviral Agent against Enterovirus A71 (EV-A71)

2021 ◽  
Vol 22 (16) ◽  
pp. 8757
Author(s):  
Salima Lalani ◽  
Malihe Masomian ◽  
Chit Laa Poh
Keyword(s):  
Foot And Mouth Disease ◽  
Antiviral Agent ◽  
Underlying Mechanism ◽  
Neurological Complications ◽  
In Vivo Evaluation ◽  
Competitive Binding Assay ◽  
Enterovirus A71 ◽  
Viral Protein 1

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.

scholarly journals Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

2007 ◽  
Vol 130 (2) ◽  
pp. 157-168 ◽  
Author(s):  
David M. Blodgett ◽  
Julie K. De Zutter ◽  
Kara B. Levine ◽  
Pusha Karim ◽  
Anthony Carruthers
Keyword(s):  
Glucose Transport ◽  
Conformational Changes ◽  
Covalent Modification ◽  
Antibody Binding ◽  
Mass Spectrometry Analysis ◽  
Structural Basis ◽  
Ionization Mass ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Lysine Residues

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7.

scholarly journals Xiao Yao San Improves Depressive-Like Behavior in Rats through Modulation ofβ-Arrestin 2-Mediated Pathways in Hippocampus

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Xiaoxia Zhu ◽  
Oudong Xia ◽  
Weili Han ◽  
Meng Shao ◽  
Linlin Jing ◽  
...  
Keyword(s):  
Social Isolation ◽  
High Performance ◽  
Mammalian Target Of Rapamycin ◽  
Underlying Mechanism ◽  
Mass Spectrometry Analysis ◽  
Mild Stress ◽  
Spectrometry Analysis ◽  
Phosphatase 2A ◽  
Classical Chinese Medicine ◽  
Tyrosine Receptor Kinase

Xiao Yao San (XYS) is a classical Chinese medicine formula that has been widely used to treat mood disorders for hundreds of years. To confirm the effect of XYS and better understand its underlying mechanism, high-performance liquid chromatography-mass spectrometry analysis-based quality control of XYS extracts and proteomics-based identification of differential proteins in the hippocampus were adopted in social isolation and chronic unpredictable mild stress- (CUMS-) treated rats. The depressive-like behavior of rats induced by CUMS resembled the manifestation of human depression. The upregulated corticosterone (CORT) and urocortin 2 (UCN2) levels demonstrated the existence of hypothalamic-pituitary-adrenal (HPA) axis hyperactivity. XYS was effective in ameliorating the depressive-like behavior and downregulating UCN2 and CORT. XYS decreased the expression of serine/threonine-protein phosphatase 2A subunit B and increased the expression ofβ-arrestin 2. The expressions of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB), and mammalian target of rapamycin (mTOR) were also elevated by XYS. In conclusion, XYS improves social isolation and CUMS-induced depressive-like behavior and ameliorates HPA hyperactivation through the downregulation of corticotrophin releasing hormone (CRH) receptor 2. The upregulation of BDNF/TrkB and the phosphorylation of mTOR requireβ-arrestin 2 as a scaffold to regulate stress signaling.

scholarly journals Characterization of A Novel Autoimmune Encephalitis Associated Antibody against CRMP2

2021 ◽  
Author(s):  
Kaibiao Xu ◽  
Dongmei Wang ◽  
Yan He ◽  
Shengnan Wang ◽  
Guanghui Liu ◽  
...  
Keyword(s):  
Rat Brain ◽  
Hek293t Cell ◽  
Autoimmune Encephalitis ◽  
Mass Spectrometry Analysis ◽  
Synaptic Protein ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Mediator Protein ◽  
Large Category

ABSTRACTObjective Autoimmune encephalitis (AE) is a large category disorder urging antibody characterization. The aim was to identify a novel AE related autoantibody targeting an intracellular synaptic protein. Methods Suspected AE patients had negative conventional antibodies screening but strong immunolabel signals on rat brain sections with the serum and cerebrospinal fluid (CSF) samples were considered burdening unkown antibody. Immunoprecipitation from the rat brain protein lysate followed by mass spectrometry analysis was used to identify the targeting antigen. Western blotting and/or cell-based assay (CBA) with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope and IgG subtype determination. Patients with similar immunostaining pattern on rat brain sections were retrospectively screened for the antibody. Results The antibody against collapsin response mediator protein 2 (CRMP2), a synaptic protein involved in axon guidance, was identified in a patient with suspected AE. The patient samples reactivated with HEK293T cell overexpressed CRMP2, rather than CRMP1, 3, 4, and 5. The patient samples mainly stained neuronal cytoplasm of the cortex, hippocampus and cerebellum Purkinje cells. This reactivity was eliminated by pre-immunoabsorption with CRMP2-overexpressing HEK293T cells. CRMP2 truncation experiments indicated that 536 amino acids at C-terminus was necessary for the epitope. Subtype analysis showed that anti-CRMP2 antibody was IgG4. Moreover, screening from 19 suspected AE patients led to identification of anti-CRMP2 antibody in another patient with a diagnosis of encephalomyelitis. The two patients responded to immunotherapy. Conclusions This study discovered a novel anti-CRMP2 antibody associated with AE. Testing of the antibody might be promising for AE diagnosis and treatment.

scholarly journals Investigation of the In-Vivo Cytotoxicity and the In Silico-Prediction of MDM2-p53 Inhibitor Potential of Euphorbia peplus Methanolic Extract in Rats

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 642 ◽  
Author(s):  
Yasmina M. Abd-Elhakim ◽  
Mohamed Abdo Nassan ◽  
Gamal A. Salem ◽  
Abdelkarim Sasi ◽  
Adil Aldhahrani ◽  
...  
Keyword(s):  
In Silico ◽  
Methanolic Extract ◽  
P53 Protein ◽  
Underlying Mechanism ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
P53 Expression ◽  
Spectrometry Analysis ◽  
Euphorbia Peplus

This study explored the probable in vivo cardiac and renal toxicities together with in silico approaches for predicting the apoptogenic potential of Euphorbia peplus methanolic extract (EPME) in rats. Cardiac and renal injury biomarkers were estimated with histopathological and immunohistochemical evaluations of both kidney and heart. The probable underlying mechanism of E. peplus compounds to potentiate p53 activity is examined using Molecular Operating Environment (MOE) docking software and validated experimentally by immunohistochemical localization of p53 protein in the kidney and heart tissues. The gas chromatography/mass spectrometry analysis of E. peplus revealed the presence of nine different compounds dominated by di-(2-ethylhexyl) phthalate (DEHP). Significant elevations of troponin, creatine phosphokinase, creatine kinase–myocardium bound, lactate dehydrogenase, aspartate transaminase, alkaline phosphatase, urea, creatinine, and uric acid were evident in the EPME treated rats. The EPME treated rats showed strong renal and cardiac p53 expression and moderate cardiac TNF-α expression. Further, our in silico results predicted the higher affinity and good inhibition of DEHP, glyceryl linolenate, and lucenin 2 to the MDM2-p53 interface compared to the standard reference 15 a compound. Conclusively, EPME long-term exposure could adversely affect the cardiac and renal tissues probably due to their inflammatory and apoptotic activity. Moreover, the in silico study hypothesizes that EPME inhibits MDM2-mediated degradation of p53 suggesting possible anticancer potentials which confirmed experimental by strong p53 expression in renal and cardiac tissues.

scholarly journals Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

2020 ◽  
Author(s):  
Onyekachi E. Ononye ◽  
Christopher W. Sausen ◽  
Lata Balakrishnan ◽  
Matthew L. Bochman
Keyword(s):  
Limited Proteolysis ◽  
Strand Break ◽  
Mass Spectrometry Analysis ◽  
Helicase Domain ◽  
Lysine Acetylation ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Substrate Preferences ◽  
C Terminus ◽  
N Terminus

ABSTRACTIn S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

scholarly journals HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Chen Li ◽  
Lin Shi ◽  
Yan Gao ◽  
Yuanan Lu ◽  
Jing Ye ◽  
...  
Keyword(s):  
G Protein ◽  
Defense Mechanism ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Lysosomal Degradation ◽  
Spectrometry Analysis ◽  
Glycoprotein G ◽  
Multicomponent Complex ◽  
Host Defense Mechanism ◽  
Novel Host

As a fierce pathogen, spring viremia of carp virus (SVCV) can cause high mortality in the common carp, and its glycoprotein (G protein) is a component of the viral structure on the surface of virion, which is crucial in viral life cycle. This report adopted tandem affinity purification (TAP), mass spectrometry analysis (LC-MS/MS), immunoprecipitation, and confocal microscopy assays to identify Heat shock cognate protein 70 (HSC70) as an interaction partner of SVCV G protein. It was found that HSC70 overexpression dramatically inhibited SVCV replication, whereas its loss of functions elicited opposing effects on SVCV replication. Mechanistic studies indicate that HSC70 induces lysosomal degradation of ubiquitinated-SVCV G protein. This study further demonstrates that Membrane-associated RING-CH 8 (MARCH8), an E3 ubiquitin ligase, is critical for SVCV G protein ubiquitylation and leads to its lysosomal degradation. Furthermore, the MARCH8 mediated ubiquitylation of SVCV G protein required the participation of HSC70 through forming a multicomponent complex. Taken together, these results demonstrate that HSC70 serves as a scaffold for MARCH8 and SVCV G, which leads to the ubiquitylation and degradation of SVCV G protein and thus inhibits viral replication. These findings have established a novel host defense mechanism against SVCV.

scholarly journals Exposing the Secrets of Two Well-Known Lactobacillus casei Phages, J-1 and PL-1, by Genomic and Structural Analysis

2014 ◽  
Vol 80 (22) ◽  
pp. 7107-7121 ◽  
Author(s):  
Maria Eugenia Dieterle ◽  
Charles Bowman ◽  
Carlos Batthyany ◽  
Esteban Lanzarotti ◽  
Adrián Turjanski ◽  
...  
Keyword(s):  
Lactobacillus Casei ◽  
Sequence Similarity ◽  
Proteolytic Processing ◽  
Mass Spectrometry Analysis ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Related Phage ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTBacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult usingLactobacillus caseistrain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in otherSiphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically toLactobacillus casei/paracaseicells, and the addition ofl-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls ofL. caseiATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into howLactobacillusphages interact with their hosts at the first steps of infection.

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