scholarly journals A novel high-throughput screen for identifying lipids that stabilise membrane proteins in detergent based solution

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254118
Author(s):  
Cristina Cecchetti ◽  
Jannik Strauss ◽  
Claudia Stohrer ◽  
Claire Naylor ◽  
Edward Pryor ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
High Throughput ◽  
Structural Integrity ◽  
Stability Assessment ◽  
Biological Functions ◽  
Starting Point ◽  
Potential Applications ◽  
Protein Research ◽  
Cholesteryl Hemisuccinate

Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A2AR). A2AR is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.

Coarse-grained simulation: a high-throughput computational approach to membrane proteins

2008 ◽  
Vol 36 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Mark S.P. Sansom ◽  
Kathryn A. Scott ◽  
Peter J. Bond
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
High Throughput ◽  
Protein Interactions ◽  
Coarse Grained ◽  
Atomistic Simulations ◽  
Lactose Permease ◽  
Coarse Grained Simulations ◽  
Dynamics Simulations ◽  
Good Agreement

An understanding of the interactions of membrane proteins with a lipid bilayer environment is central to relating their structure to their function and stability. A high-throughput approach to prediction of membrane protein interactions with a lipid bilayer based on coarse-grained Molecular Dynamics simulations is described. This method has been used to develop a database of CG simulations (coarse-grained simulations) of membrane proteins (http://sbcb.bioch.ox.ac.uk/cgdb). Comparison of CG simulations and AT simulations (atomistic simulations) of lactose permease reveals good agreement between the two methods in terms of predicted lipid headgroup contacts. Both CG and AT simulations predict considerable local bilayer deformation by the voltage sensor domain of the potassium channel KvAP.

tiRNAs & tRFs Biogenesis and Regulation of Diseases: A Review

2019 ◽  
Vol 26 (31) ◽  
pp. 5849-5861 ◽  
Author(s):  
Pan Jiang ◽  
Feng Yan
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Metabolic Diseases ◽  
Viral Infections ◽  
Regulatory Mechanisms ◽  
Biological Functions ◽  
Reverse Transcriptases ◽  
Dna Damage Responses ◽  
Potential Applications ◽  
Viral Reverse Transcriptases

tiRNAs & tRFs are a class of small molecular noncoding tRNA derived from precise processing of mature or precursor tRNAs. Most tiRNAs & tRFs described originate from nucleus-encoded tRNAs, and only a few tiRNAs and tRFs have been reported. They have been suggested to play important roles in inhibiting protein synthesis, regulating gene expression, priming viral reverse transcriptases, and the modulation of DNA damage responses. However, the regulatory mechanisms and potential function of tiRNAs & tRFs remain poorly understood. This review aims to describe tiRNAs & tRFs, including their structure, biological functions and subcellular localization. The regulatory roles of tiRNAs & tRFs in translation, neurodegeneration, metabolic diseases, viral infections, and carcinogenesis are also discussed in detail. Finally, the potential applications of these noncoding tRNAs as biomarkers and gene regulators in different diseases is also highlighted.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

scholarly journals Fungal Melanins and Applications in Healthcare, Bioremediation and Industry

2021 ◽  
Vol 7 (6) ◽  
pp. 488
Author(s):  
Ellie Rose Mattoon ◽  
Radames J. B. Cordero ◽  
Arturo Casadevall
Keyword(s):  
Chemical Structure ◽  
Industrial Processes ◽  
Metal Chelation ◽  
Biological Functions ◽  
Societal Benefit ◽  
Potential Applications ◽  
Fungal Melanin ◽  
Human Use ◽  
Fungal Melanins ◽  
Isolated Species

Melanin is a complex multifunctional pigment found in all kingdoms of life, including fungi. The complex chemical structure of fungal melanins, yet to be fully elucidated, lends them multiple unique functions ranging from radioprotection and antioxidant activity to heavy metal chelation and organic compound absorption. Given their many biological functions, fungal melanins present many possibilities as natural compounds that could be exploited for human use. This review summarizes the current discourse and attempts to apply fungal melanin to enhance human health, remove pollutants from ecosystems, and streamline industrial processes. While the potential applications of fungal melanins are often discussed in the scientific community, they are successfully executed less often. Some of the challenges in the applications of fungal melanin to technology include the knowledge gap about their detailed structure, difficulties in isolating melanotic fungi, challenges in extracting melanin from isolated species, and the pathogenicity concerns that accompany working with live melanotic fungi. With proper acknowledgment of these challenges, fungal melanin holds great potential for societal benefit in the coming years.

scholarly journals Lipid Transporters Beam Signals from Cell Membranes

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 562
Author(s):  
Miliça Ristovski ◽  
Danny Farhat ◽  
Shelly Ellaine M. Bancud ◽  
Jyh-Yeuan Lee
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Lipid Composition ◽  
Structural Integrity ◽  
Current Knowledge ◽  
Integral Membrane Proteins ◽  
Cellular Membranes ◽  
Cytoplasmic Proteins ◽  
Lipid Transporters

Lipid composition in cellular membranes plays an important role in maintaining the structural integrity of cells and in regulating cellular signaling that controls functions of both membrane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transporters, two integral membrane proteins, are known to contribute to lipid translocation across the lipid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membranes in regulating cell signaling and how lipid transporters participate this process.

Acoustic Emission Monitoring of Leaks in Pipes for Transport of Liquid and Gaseous Media: A Model Experiment

2006 ◽  
Vol 13-14 ◽  
pp. 351-356 ◽  
Author(s):  
Andreas J. Brunner ◽  
Michel Barbezat
Keyword(s):  
Acoustic Emission ◽  
Model Experiment ◽  
Structural Integrity ◽  
Fiber Composite ◽  
Leak Testing ◽  
Pipe Segment ◽  
Potential Applications ◽  
Gaseous Media ◽  
The Media ◽  
Ae Signals

In order to explore potential applications for Active Fiber Composite (AFC) elements made from piezoelectric fibers for structural integrity monitoring, a model experiment for leak testing on pipe segments has been designed. A pipe segment made of aluminum with a diameter of 60 mm has been operated with gaseous (compressed air) and liquid media (water) for a range of operating pressures (between about 5 and 8 bar). Artificial leaks of various sizes (diameter) have been introduced. In the preliminary experiments presented here, commercial Acoustic Emission (AE) sensors have been used instead of the AFC elements. AE sensors mounted on waveguides in three different locations have monitored the flow of the media with and without leaks. AE signals and AE waveforms have been recorded and analysed for media flow with pressures ranging from about 5 to about 8 bar. The experiments to date show distinct differences in the FFT spectra depending on whether a leak is present or not.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

scholarly journals Hydrodynamic Boundary Layers at Solid Wall—A Tool for Separation of Fine Solids

2021 ◽  
Vol 5 (1) ◽  
pp. 11
Author(s):  
Ljubomir Nikolov
Keyword(s):  
Relative Density ◽  
Hydrodynamic Interaction ◽  
Theoretical Study ◽  
Solid Wall ◽  
Separation Processes ◽  
Starting Point ◽  
Potential Applications ◽  
Neutrally Buoyant ◽  
Solid Walls ◽  
Solid Spheres

A theoretical study is performed about the hydrodynamic interaction of fine species entrapped in the boundary layer (BL) at solid wall (plate). The key starting point is the analysis of the disturbance introduced by solid spheres in the background fluid flow. For a neutrally buoyant entity, the type of interaction is determined by the size of the spheres as compared to the thickness of the BL region. The result is granulometric separation of the solids inside the BL domain at the wall. The most important result in view of potential applications concerns the so-called small particles Rp < L/ReL5/4 (ReL is the Reynolds number of the background flow and Rp is the radius of the entrapped sphere). In the case of non-neutrally buoyant particles, gravity interferes with the separation effect. Important factor in this case is the relative density of the solid species as compared to this of the fluid. In view of further practical uses, particles within the range of Δρ/ρ < Fr2/ReL1/2 (Fr is Froude number and Δρ/ρ is the relative density of the entrapped solids) are systematically studied. The trajectories inside the BL region of the captured species are calculated. The obtained data show that there are preferred regions along the wall where the fine solids are detained. The results are important for the assessment of the general efficiency of entrapment and segregation of fine species in the vicinity of solid walls and have high potential for further design of industrial separation processes.

Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay

2016 ◽  
Vol 21 (10) ◽  
pp. 1042-1053 ◽  
Author(s):  
Clara Stead ◽  
Adam Brown ◽  
Cathryn Adams ◽  
Sarah J. Nickolls ◽  
Gareth Young ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
Glycine Receptor ◽  
Functional Characterization ◽  
In Vitro Assays ◽  
Allosteric Modulators ◽  
Inhibitory Neurotransmission ◽  
Starting Point ◽  
Positive Allosteric Modulators

Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent membrane potential screen and a medium-throughput electrophysiology assay using IonFlux HT together with conventional manual patch clamp. Using these assays, we conducted a primary screening campaign and report the structures of hit compounds identified as GlyR PAMs. Our functional characterization data reveal a hit compound with high efficacy relative to current known potentiators and selectivity over GABAAR, another major class of inhibitory neurotransmission receptors of importance to pain. These small-molecule GlyR PAMs have high potential both as early tool compounds to enable pharmacological studies of GlyR inhibitory neurotransmission and as a starting point for the development of potent, selective GlyRα3 PAMs as novel analgesics.

Calcium-induced reversible assembly of phosphorylated amphiphile within lipid bilayer membranes

2021 ◽  
Author(s):  
Yusuke Shimizu ◽  
Kohei Sato ◽  
Kazushi Kinbara
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Phenylene Ethynylene ◽  
Lipid Bilayer Membranes ◽  
Bilayer Membranes

Inspired by calcium-induced reversible assembly and disassembly of membrane proteins found in nature, here we developed a phosphorylated amphiphile (PA) that contains an oligo(phenylene-ethynylene) unit as a hydrophobic unit and...

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