Using FRET to measure the time it takes for a cell to destroy a virus

Nanoscale ◽  
2020 ◽  
Vol 12 (16) ◽  
pp. 9124-9132 ◽  
Author(s):  
Candace E. Benjamin ◽  
Zhuo Chen ◽  
Olivia R. Brohlin ◽  
Hamilton Lee ◽  
Arezoo Shahrivarkevishahi ◽  
...  
Keyword(s):  
A Cell ◽  
Viral Nanoparticles ◽  
Open Question ◽  
In Cells

The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question.

Using FRET to Measure the Time it Takes for a Cell to Destroy a Virus

2019 ◽  
Author(s):  
Candace E. Benjamin ◽  
Zhuo Chen ◽  
Olivia Brohlin ◽  
Hamilton Lee ◽  
Stefanie Boyd ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Rhodamine B ◽  
Virus Like Particle ◽  
A Cell ◽  
Viral Nanoparticles ◽  
The Stability ◽  
Open Question ◽  
Viral Surface ◽  
Fret Pair

<div><div><div><p>The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qβ - a representative and popular VLP for several applications - following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle’s behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.</p></div></div></div>

Using FRET to Measure the Time it Takes for a Cell to Destroy a Virus

2019 ◽  
Author(s):  
Candace E. Benjamin ◽  
Zhuo Chen ◽  
Olivia Brohlin ◽  
Hamilton Lee ◽  
Stefanie Boyd ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Rhodamine B ◽  
Virus Like Particle ◽  
A Cell ◽  
Viral Nanoparticles ◽  
The Stability ◽  
Open Question ◽  
Viral Surface ◽  
Fret Pair

<div><div><div><p>The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qβ - a representative and popular VLP for several applications - following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle’s behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.</p></div></div></div>

scholarly journals PTP4A3, A Novel Target Gene of HIF-1alpha, Participates in Benzene-Induced Cell Proliferation Inhibition and Apoptosis through PI3K/AKT Pathway

2020 ◽  
Vol 17 (3) ◽  
pp. 910
Author(s):  
Yunqiu Pu ◽  
Fengxia Sun ◽  
Rongli Sun ◽  
Zhaodi Man ◽  
Shuangbin Ji ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Target Gene ◽  
Akt Pathway ◽  
Proliferation Inhibition ◽  
Cell Proliferation Inhibition ◽  
A Cell ◽  
Novel Target ◽  
In Cells

Benzene, a commonly used chemical, has been confirmed to specifically affect the hematopoietic system as well as overall human health. PTP4A3 is overexpressed in leukemia cells and is related to cell proliferation. We previously found that HIF-1alpha was involved in benzene toxicity and PTP4A3 may be the target gene of HIF-1alpha via ChIP-seq. The aim of this study is to confirm the relationship between HIF-1alpha and PTP4A3 in benzene toxicity, as well as the function of PTP4A3 on cell toxicity induced by 1,4-benzoquinone (1,4-BQ). Our results indicate that HIF-1alpha could regulate PTP4A3 with in vivo and in vitro experiments. A cell line with suppressed PTP4A3 was established to investigate the function of PTP4A3 in 1,4-BQ toxicity in vitro. The results revealed that cell proliferation inhibition was more aggravated in PTP4A3 low-expression cells than in the control cells after 1,4-BQ treatment. The relative oxygen species (ROS) significantly increased in cells with inhibited PTP4A3, while the rise was inferior to the control cells at the 20 μM 1,4-BQ group. An increase in DNA damage was seen in PTP4A3 down-regulated cells at the 10 μM 1,4-BQ group, whereas the results reversed at the concentration of 20 μM. Moreover, the apoptosis rate increased higher in down-regulated PTP4A3 cells after 1,4-BQ exposure. In addition, PI3K/AKT pathway was significantly restrained in cells with inhibited PTP4A3 after 1,4-BQ treatment. Our results indicate that HIF-1alpha may regulate PTP4A3 to be involved in benzene toxicity. Inhibition of PTP4A3 could aggravate cell proliferation suppression and apoptosis by regulating PI3K/AKT pathway after 1,4-BQ treatment.

scholarly journals A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Miyuki Suzawa ◽  
Diego A Miranda ◽  
Karmela A Ramos ◽  
Kenny K-H Ang ◽  
Emily J Faivre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Toxic Chemical ◽  
Primary Hepatocytes ◽  
Humanized Mouse ◽  
Post Translational Modifications ◽  
Sumo Modification ◽  
A Cell ◽  
Primary Gene ◽  
In Cells

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.

scholarly journals The J domain of sacsin disrupts intermediate filament assembly

2021 ◽  
Author(s):  
Afrooz Dabbaghizadeh ◽  
Alexandre Pare ◽  
Zacharie Cheng-Boivin ◽  
Robin Dagher ◽  
Sandra Minotti ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Loss Of Function ◽  
Cultured Fibroblasts ◽  
Filament Assembly ◽  
A Cell ◽  
J Domain ◽  
Initial Assembly ◽  
In Cells

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS), is caused by loss of function mutations in the SACS gene, which encodes sacsin, a giant protein of 520 kDa. A key feature of the absence of sacsin in cells is the formation of abnormal bundles of intermediate filaments (IF) including neurofilaments (NF) in neurons and vimentin IF in fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons, however, its mechanism is still unclear. Here, we focused on the role of SacsJ in NF assembly. We report that the SacsJ domain directly interacts with NF proteins in vitro to disassemble NFL filaments, and to inhibit their initial assembly, in the absence of Hsp70. We generated a cell-penetrating peptide derived from this domain, SacsJ-myc-TAT, which was efficient in disassembling both vimentin IF and NF in cultured fibroblasts and Sacs+/+ motor neurons as well as NF bundles in cultured Sacs-/- motor neurons. Whereas a normal NF network was restored in Sacs-/- neurons treated with the SacsJ peptide, there was some loss of IF networks in Sacs+/+ fibroblasts or neurons. These results suggest that SacsJ is a key regulator of NF and IF networks in cells, with implications for its therapeutic use.

scholarly journals Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.

1996 ◽  
Vol 184 (4) ◽  
pp. 1331-1341 ◽  
Author(s):  
S A Susin ◽  
N Zamzami ◽  
M Castedo ◽  
T Hirsch ◽  
P Marchetti ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Chromatin Condensation ◽  
Interleukin 1 ◽  
Permeability Transition ◽  
Mitochondrial Inner Membrane ◽  
Nuclear Changes ◽  
A Cell ◽  
Apoptosis Inducing Factor ◽  
In Cells

Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.

scholarly journals Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures duringListeriaInfections

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Aaron S. Dhanda ◽  
A. Wayne Vogl ◽  
Sharifah E. Albraiki ◽  
Carol A. Otey ◽  
Moriah R. Beck ◽  
...  
Keyword(s):  
Cell Line ◽  
Structural Integrity ◽  
Comet Tail ◽  
Null Cell ◽  
Free System ◽  
Content Type ◽  
A Cell ◽  
Protein Components ◽  
In Cells

ABSTRACTPalladin is an important component of motile actin-rich structures and nucleates branched actin filament arraysin vitro. Here we examine the role of palladin duringListeria monocytogenesinfections in order to tease out novel functions of palladin. We show that palladin is co-opted byL. monocytogenesduring its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells,L. monocytogenesfailed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability ofL. monocytogenesto generate comet tails was restored. Using purified protein components, we demonstrate thatL. monocytogenesactin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.IMPORTANCEStructures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made byListeria. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.

scholarly journals A cell-penetrating protein designed for bimodal fluorescence and magnetic resonance imaging

2015 ◽  
Vol 6 (11) ◽  
pp. 6607-6613 ◽  
Author(s):  
Qin Wu ◽  
Qinqin Cheng ◽  
Siming Yuan ◽  
Junchao Qian ◽  
Kai Zhong ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Magnetic Resonance ◽  
Resonance Imaging ◽  
Imaging Probe ◽  
Bimodal Imaging ◽  
Cell Penetrating ◽  
A Cell ◽  
In Cells

A protein-based fluorescence and magnetic resonance bimodal imaging probe was explored in vitro, in cells and in mice.

scholarly journals Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress responses

2018 ◽  
Author(s):  
Magdalena M Szewczyk ◽  
Yoshinori Ishikawa ◽  
Shawna Organ ◽  
Nozomu Sakai ◽  
Fengling Li ◽  
...  
Keyword(s):  
Stress Responses ◽  
Drug Targets ◽  
Proteasome Inhibitors ◽  
Arginine Methylation ◽  
Protein Arginine Methyltransferases ◽  
Hsp70 Family ◽  
Associated Proteins ◽  
A Cell ◽  
In Cells

AbstractProtein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective and cell active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells, is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 resulted in drastically reduced levels of arginine monomethylation of HSP70 family members and other stress-associated proteins. Structural and biochemical analysis revealed that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibited methylation of both constitutive and inducible forms of HSP70, and led to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

scholarly journals Novel Dengue Virus NS2B/NS3 Protease Inhibitors

2014 ◽  
Vol 59 (2) ◽  
pp. 1100-1109 ◽  
Author(s):  
Hongmei Wu ◽  
Stefanie Bock ◽  
Mariya Snitko ◽  
Thilo Berger ◽  
Thomas Weidner ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rational Drug Design ◽  
Denv Serotypes ◽  
Benzothiazole Derivatives ◽  
Rational Drug ◽  
A Cell ◽  
The Moment ◽  
Ns3 Protease ◽  
In Cells

ABSTRACTDengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PRin vitroand in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to thein vitroassays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.

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