scholarly journals Simvastatin Efficiently Reduces Levels of Alzheimer’s Amyloid Beta in Yeast

2019 ◽  
Vol 20 (14) ◽  
pp. 3531 ◽  
Author(s):  
Sudip Dhakal ◽  
Mishal Subhan ◽  
Joshua M. Fraser ◽  
Kenneth Gardiner ◽  
Ian Macreadie
Keyword(s):  
Fusion Protein ◽  
Amyloid Beta ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Dependent Manner ◽  
Green Fluorescence ◽  
Convenient Means ◽  
Ergosterol Synthesis ◽  
Green Fluorescent

A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism, Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

scholarly journals Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

2014 ◽  
Vol 19 (2) ◽  
pp. 215-222 ◽  
Author(s):  
Simon J. Gruber ◽  
Razvan L. Cornea ◽  
Ji Li ◽  
Kurt C. Peterson ◽  
Tory M. Schaaf ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Chemical Library ◽  
Time Resolved ◽  
Endoplasmic Reticulum Calcium ◽  
Green Fluorescent

We have used a “two-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

scholarly journals Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (6) ◽  
pp. 945-953 ◽  
Author(s):  
Guido Grossmann ◽  
Miroslava Opekarova ◽  
Linda Novakova ◽  
Jürgen Stolz ◽  
Widmar Tanner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Fusion Protein ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Gfp Fusion Protein ◽  
Ergosterol Synthesis ◽  
Membrane Compartments ◽  
Green Fluorescent

ABSTRACT The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H+ symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H+/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.

scholarly journals Arabidopsis COPPER TRANSPORTER 1 undergoes degradation in a proteasome-dependent manner

2020 ◽  
Vol 71 (19) ◽  
pp. 6174-6186
Author(s):  
Jinjin Li ◽  
Jinhong Yuan ◽  
Hui Wang ◽  
Hui Zhang ◽  
Haiyan Zhang
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Copper Uptake ◽  
Atpase Inhibitor ◽  
Copper Transporter ◽  
Excess Copper ◽  
Trafficking Inhibitor ◽  
Green Fluorescent

Abstract The essential nutrient copper is toxic in excess. Therefore, plants must tightly control copper uptake and distribution. Arabidopsis thaliana high-affinity copper transporters (COPTs) mediate copper uptake, partitioning, and redistribution. Here we show that COPT1 localizes to the plasma membrane and endoplasmic reticulum in stably transgenic plants expressing a COPT1–green fluorescent protein (GFP) fusion protein, and the fusion protein is rapidly degraded upon plant exposure to excess copper. MG132 treatment largely abolished copper-induced degradation of COPT1, implying a link between the proteasome and COPT1 activity in modulating copper uptake. Co-immunoprecipitation analyses revealed that COPT1 cannot be ubiquitinated in the presence of excess copper and MG132. Through site-directed mutagenesis, we identified Lys159 in the C-terminal cytoplasmic tail of COPT1 as critical for copper acquisition, but not for copper-mediated down-regulation of COPT1, in plants. Furthermore, pharmacological analysis showed that treatment with a vesicle trafficking inhibitor or a V-ATPase inhibitor does not alter the subcellular dynamics of COPT1–GFP, consistent with the absence of a connection between the endosomal recycling/vacuolar system and COPT1 degradation. Together, our data suggest that proteasomal degradation rather than vacuolar proteolysis is important for the regulation of copper transport to maintain copper homeostasis in plants.

scholarly journals A Toxic Synergy between Aluminium and Amyloid Beta in Yeast

2021 ◽  
Vol 22 (4) ◽  
pp. 1835
Author(s):  
Jamieson B. Mcdonald ◽  
Sudip Dhakal ◽  
Ian Macreadie
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Growth Inhibition ◽  
Cell Viability ◽  
Amyloid Beta ◽  
Fluorescent Protein ◽  
Late Onset ◽  
Model Organism ◽  
Age Related ◽  
Green Fluorescent

Alzheimer’s disease (AD), the most prevalent, age-related, neurodegenerative disease, is associated with the accumulation of amyloid beta (Aβ) and oxidative stress. However, the sporadic nature of late-onset AD has suggested that other factors, such as aluminium may be involved. Aluminium (Al3+) is the most ubiquitous neurotoxic metal on earth, extensively bioavailable to humans. Despite this, the link between Al3+ and AD has been debated for decades and remains controversial. Using Saccharomyces cerevisiae as a model organism expressing Aβ42, this study aimed to examine the mechanisms of Al3+ toxicity and its interactions with Aβ42. S. cerevisiae cells producing Aβ42 treated with varying concentrations of Al3+ were examined for cell viability, growth inhibition, and production of reactive oxygen species (ROS). Al3+ caused a significant reduction in cell viability: cell death in yeast producing green fluorescent protein tagged with Aβ42 (GFP–Aβ42) was significantly higher than in cells producing green fluorescent protein (GFP) alone. Additionally, Al3+ greatly inhibited the fermentative growth of yeast producing GFP–Aβ42, which was enhanced by ferric iron (Fe3+), while there was negligible growth inhibition of GFP cells. Al3+- induced ROS levels in yeast expressing native Aβ42 were significantly higher than in empty vector controls. These findings demonstrate Al3+ has a direct, detrimental toxic synergy with Aβ42 that can be influenced by Fe3+, causing increased oxidative stress. Thus, Al3+ should be considered as an important factor, alongside the known characteristic hallmarks of AD, in the development and aetiology of the disease.

scholarly journals Alteration of Large-Scale Chromatin Structure by Estrogen Receptor

2002 ◽  
Vol 22 (10) ◽  
pp. 3437-3449 ◽  
Author(s):  
Anne C. Nye ◽  
Ramji R. Rajendran ◽  
David L. Stenoien ◽  
Michael A. Mancini ◽  
Benita S. Katzenellenbogen ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Fusion Protein ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Nuclear Hormone Receptor ◽  
Live Cells ◽  
Interaction Domain ◽  
Green Fluorescent

ABSTRACT The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily important in human physiology and disease, recruits coactivators which modify local chromatin structure. Here we describe effects of ER on large-scale chromatin structure as visualized in live cells. We targeted ER to gene-amplified chromosome arms containing large numbers of lac operator sites either directly, through a lac repressor-ER fusion protein (lac rep-ER), or indirectly, by fusing lac repressor with the ER interaction domain of the coactivator steroid receptor coactivator 1. Significant decondensation of large-scale chromatin structure, comparable to that produced by the ∼150-fold-stronger viral protein 16 (VP16) transcriptional activator, was produced by ER in the absence of estradiol using both approaches. Addition of estradiol induced a partial reversal of this unfolding by green fluorescent protein-lac rep-ER but not by wild-type ER recruited by a lac repressor-SRC570-780 fusion protein. The chromatin decondensation activity did not require transcriptional activation by ER nor did it require ligand-induced coactivator interactions, and unfolding did not correlate with histone hyperacetylation. Ligand-induced coactivator interactions with helix 12 of ER were necessary for the partial refolding of chromatin in response to estradiol using the lac rep-ER tethering system. This work demonstrates that when tethered or recruited to DNA, ER possesses a novel large-scale chromatin unfolding activity.

scholarly journals A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency

2021 ◽  
pp. 247255522110181
Author(s):  
Andreas Vogt ◽  
Samantha L. Eicher ◽  
Tracey D. Myers ◽  
Stacy L. Hrizo ◽  
Laura L. Vollmer ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Triose Phosphate Isomerase ◽  
Pharmacological Intervention ◽  
Screening Assay ◽  
Triose Phosphate ◽  
The Common ◽  
Green Fluorescent

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the “common” TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose–response, by limited structure–activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

scholarly journals Protection against Autoimmune Diabetes by Silkworm-Produced GFP-Tagged CTB-Insulin Fusion Protein

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Qiaohong Meng ◽  
Wenfeng Wang ◽  
Xiaowen Shi ◽  
Yongfeng Jin ◽  
Yaozhou Zhang
Keyword(s):  
Fusion Protein ◽  
Oral Administration ◽  
Fluorescent Protein ◽  
Autoimmune Diabetes ◽  
Nod Mice ◽  
Treg Cells ◽  
Immunological Tolerance ◽  
Protein Vaccine ◽  
Green Fluorescent

In animals, oral administration of the cholera toxin B (CTB) subunit conjugated to the autoantigen insulin enhances the specific immune-unresponsive state. This is called oral tolerance and is capable of suppressing autoimmune type 1 diabetes (T1D). However, the process by which the CTB-insulin (CTB-INS) protein works as a therapy for T1Din vivoremains unclear. Here, we successfully expressed a green fluorescent protein- (GFP-) tagged CTB-Ins (CTB-Ins-GFP) fusion protein in silkworms in a pentameric form that retained the native ability to activate the mechanism. Oral administration of the CTB-Ins-GFP protein induced special tolerance, delayed the development of diabetic symptoms, and suppressed T1D onset in nonobese diabetic (NOD) mice. Moreover, it increased the numbers of CD4+CD25+Foxp3+T regulatory (Treg) cells in peripheral lymph tissues and affected the biological activity of spleen cells. This study demonstrated that the CTB-Ins-GFP protein produced in silkworms acted as an oral protein vaccine, inducing immunological tolerance involving CD4+CD25+Foxp3+Treg cells in treating T1D.

The Fe-Core/Carbon-Shell Ultrafine Nanopowders as Platform for Biomolecules Grafting

2014 ◽  
Vol 1040 ◽  
pp. 194-198
Author(s):  
N.S. Surgutskaya ◽  
P.S. Postnikov ◽  
Alexandra G. Pershina ◽  
A.I. Galanov ◽  
Marina E. Trusova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Carbon Shell ◽  
Shell Nanoparticles ◽  
Powder Surface ◽  
Metal Precursors ◽  
Covalent Grafting ◽  
Large Scale Synthesis ◽  
Green Fluorescent

The Fe-core/carbon-shell nanopowders are excellent platform for covalent grafting of biomolecules. The large-scale synthesis of Fe-core/carbon-shell nanoparticles via electropulse erosion of metal precursors in hydrocarbons was developed. The green fluorescent protein was covalently attached to the powder surface via diazonium functionalization and further carbodiimide activation.

scholarly journals Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

2020 ◽  
Vol 22 (1) ◽  
pp. 90
Author(s):  
Mehdi Kabani
Keyword(s):  
Protein Quality ◽  
Fluorescent Protein ◽  
Physical Nature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Prions ◽  
Daughter Cells ◽  
Cytoplasmic Proteins ◽  
Green Fluorescent ◽  
Functionally Diverse

The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud.

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