Critique of the use of fluorescence‐based reporters in <i>Escherichia coli</i> as a screening tool for the identification of peptide inhibitors of Aβ42 aggregation

Wright, Oliver; Liao, Zaiyi; Liu, Yun; Yoshimi, Tatsuya; Zheng, Youliang; Tunnacliffe, Alan

doi:10.1002/psc.2474

Cited by 4 publications

(3 citation statements)

References 70 publications

(129 reference statements)

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“…Nevertheless, in the recent study Wright and co-workers found that fluorescent aggregates form in nearly all cells expressing a misfolded product of exon I of Huntington gene with a N-terminal GFP reporter, while total fluorescence output per cell was equivalent to that of GFP alone. 28 This phenomenon might be explained by the differences in the folding rates of the reporter and target. If the folding rate of the target protein is slower than GFP folding time, the reporter maintains its chromophore even though the target is misfolded.…”

Section: Discussionmentioning

confidence: 99%

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Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design

et al. 2014

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Several protein expression systems for construction of protein arrays have been established in recent years. However, current protocols for protein synthesis are still time consuming, laborious and expensive. This study has established an alternative workflow that covers rapid construction of expression cassettes, in-tube and on-membrane synthesis of recombinant proteins, and straightforward screening of synthesized proteins. Eighteen membrane associated eukaryotic proteins and two secretory complement regulators (C1 inhibitor and vitronectin) were included in the study. To generate hybrid genes, double-overlap extension PCR was employed to fuse the 5' fragment (consisting of a T7 promoter and a species independent translation sequence), ORFs of the target proteins, and the 3' fragment (encompassing GFP fusion, Myc-tag and stop codon). OE-PCR generated fragments were directly mixed with the Leishmania torentolae lysate (translation mix) for protein synthesis. In order to establish a cheap and user-friendly alternative to existing cell-free protein array techniques, PCR products were spotted on the hydrophobic substrate (PVDF membrane), air-dried and covered with only 2 μL of translation mix. All synthesized proteins were spontaneously immobilized on the membrane due to the hydrophobic interaction between C-terminally fused GFP and PVDF. Synthesis and immobilization of proteins were confirmed simply by assessing the GFP chromophore under a laser scanner or a fluorescent microscope.

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Section: Discussionmentioning

confidence: 99%

“…If the folding rate of the target protein is slower than GFP folding time, the reporter maintains its chromophore even though the target is misfolded. 28 Although, the GFP reporter helps to judge the proper folding of the target protein, in some cases results should be interpreted with caution. Fig.…”

Section: Discussionmentioning

confidence: 99%

Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design

et al. 2014

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“…Indeed, a correlation of oligomer formation and stop codon read-through was confirmed by biochemical analysis 2829. An important distinction of this approach from previous anti-Aβ aggregation screens 303132 is that we can detect drugs that inhibit Aβ 42 oligomer formation but do not inhibit the formation of large Aβ 42 amyloid. This is important because such large aggregates are now thought to be helpful because they likely capture some of the more toxic Aβ 42 oligomers, rendering them less toxic 3334…”

Section: Introductionmentioning

confidence: 86%

Inhibition of Aβ42 oligomerization in yeast by a PICALM ortholog and certain FDA approved drugs

Park¹,

Ratia²,

Ba³

et al. 2016

MIC

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The formation of small Aβ42 oligomers has been implicated as a toxic species in Alzheimer disease (AD). In strong support of this hypothesis we found that overexpression of Yap1802, the yeast ortholog of the human AD risk factor, phosphatidylinositol binding clathrin assembly protein (PICALM), reduced oligomerization of Aβ42 fused to a reporter in yeast. Thus we used the Aβ42-reporter system to identify drugs that could be developed into therapies that prevent or arrest AD. From a screen of 1,200 FDA approved drugs and drug-like small compounds we identified 7 drugs that reduce Aβ42 oligomerization in yeast: 3 antipsychotics (bromperidol, haloperidol and azaperone), 2 anesthetics (pramoxine HCl and dyclonine HCl), tamoxifen citrate, and minocycline HCl. Also, all 7 drugs caused Aβ42 to be less toxic to PC12 cells and to relieve toxicity of another yeast AD model in which Aβ42 aggregates targeted to the secretory pathway are toxic. Our results identify drugs that inhibit Aβ42 oligomers from forming in yeast. It remains to be determined if these drugs inhibit Aβ42 oligomerization in mammals and could be developed as a therapeutic treatment for AD.

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Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects

Liu

Canseco-Alba

Liang

et al. 2020

IJMS

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There are two well-characterized cannabinoid receptors (CB1R and CB2R and other candidates): the central nervous system (CNS) enriched CB1R and peripheral tissue enriched CB2R with a wide dynamic range of expression levels in different cell types of human tissues. Hepatocytes and neurons express low baseline CB1R and CB2R, respectively, and their cell-type-specific functions are not well defined. Here we report inducible expression of CB1R in the liver by high-fat and high sugar diet and CB2R in cortical neurons by methamphetamine. While there is less controversy about hepatocyte CB1R, the presence of functional neuronal CB2R is still debated to date. We found that neuron CB2R basal expression was higher than that of hepatocyte CB1R by measuring mRNA levels of specific isoform CB2A in neurons isolated by fluorescence-activated cell sorting (FACS) and CB1A in hepatocytes isolated by collagenase perfusion of liver. For in vivo studies, we generated hepatocyte, dopaminergic neuron, and microglia-specific conditional knockout mice (Abl-Cnr1Δ, Dat-Cnr2Δ, and Cx3cr1-Cnr2Δ) of CB1R and CB2R by crossing Cnr1f/f and Cnr2f/f strains to Abl-Cre, Dat-Cre, and Cx3cr1-Cre deleter mouse strains, respectively. Our data reveals that neuron and microglia CB2Rs are involved in the “tetrad” effects of the mixed agonist WIN 55212-2, CB1R selective agonist arachidonyl-2′-chloroethylamide (ACEA), and CB2R selective agonist JWH133. Dat-Cnr2Δ and Cx3cr1-Cnr2Δ mice showed genotypic differences in hypomobility, hypothermia, analgesia, and catalepsy induced by the synthetic cannabinoids. Alcohol conditioned place preference was abolished in DAT-Cnr2Δ mice and remained intact in Cx3cr1-Cnr2Δ mice in comparison to WT mice. These Cre-loxP recombinant mouse lines provide unique approaches in cannabinoid research for dissecting the complex endocannabinoid system that is implicated in many chronic disorders.

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Critique of the use of fluorescence‐based reporters in Escherichia coli as a screening tool for the identification of peptide inhibitors of Aβ42 aggregation

Cited by 4 publications

References 70 publications

Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design

Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design

Inhibition of Aβ42 oligomerization in yeast by a PICALM ortholog and certain FDA approved drugs

Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects

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