scholarly journals Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: a Molecular Simulation Study

2020 ◽  
Author(s):  
Zeineb Si Chaib ◽  
Alessandro Marchetto ◽  
Klevia Dishnica ◽  
Paolo Carloni ◽  
Alejandro Giorgetti ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Translocator Protein ◽  
Coarse Grained ◽  
Mouse Protein ◽  
Two Factors ◽  
Domains Of Life ◽  
Contact Residues ◽  
The Stability

The translocator protein (TSPO) is a transmembrane protein present in the three domains of life. Its functional quaternary structure consists of one or more subunits. In mouse, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

scholarly journals Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: A Molecular Simulation Study

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4299 ◽  
Author(s):  
Zeineb Si Chaib ◽  
Alessandro Marchetto ◽  
Klevia Dishnica ◽  
Paolo Carloni ◽  
Alejandro Giorgetti ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Translocator Protein ◽  
Coarse Grained ◽  
Mouse Protein ◽  
Two Factors ◽  
Domains Of Life ◽  
Contact Residues ◽  
The Stability

The translocator protein (TSPO) is a transmembrane protein present across the three domains of life. Its functional quaternary structure consists of one or more subunits. In mice, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

scholarly journals Structural Stabilization of Human Transthyretin by Centella asiatica (L.) Urban Extract: Implications for TTR Amyloidosis

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 128 ◽  
Author(s):  
Fredrick Nwude Eze ◽  
Ladda Leelawatwattana ◽  
Porntip Prapunpoj
Keyword(s):  
Protein Misfolding ◽  
Quaternary Structure ◽  
Centella Asiatica ◽  
Structural Stabilization ◽  
Flight Mass Spectrometry ◽  
Transmission Electron ◽  
Small Molecule Ligands ◽  
Therapeutic Development ◽  
The Stability

Transthyretin is responsible for a series of highly progressive, degenerative, debilitating, and incurable protein misfolding disorders known as transthyretin (TTR) amyloidosis. Since dissociation of the homotetrameric protein to its monomers is crucial in its amyloidogenesis, stabilizing the native tetramer from dissociating using small-molecule ligands has proven a viable therapeutic strategy. The objective of this study was to determine the potential role of the medicinal herb Centella asiatica on human transthyretin (huTTR) amyloidogenesis. Thus, we investigated the stability of huTTR with or without a hydrophilic fraction of C. asiatica (CAB) against acid/urea-mediated denaturation. We also determined the influence of CAB on huTTR fibrillation using transmission electron microscopy. The potential binding interactions between CAB and huTTR was ascertained by nitroblue tetrazolium redox-cycling and 8-anilino-1-naphthalene sulfonic acid displacement assays. Additionally, the chemical profile of CAB was determined by liquid chromatography quadruple time-of-flight mass spectrometry (HPLC-QTOF-MS). Our results strongly suggest that CAB bound to and preserved the quaternary structure of huTTR in vitro. CAB also prevented transthyretin fibrillation, although aggregate formation was unmitigated. These effects could be attributable to the presence of phenolics and terpenoids in CAB. Our findings suggest that C. asiatica contains pharmaceutically relevant bioactive compounds which could be exploited for therapeutic development against TTR amyloidosis.

scholarly journals A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Markus Teucher ◽  
Hui Zhang ◽  
Verian Bader ◽  
Konstanze F. Winklhofer ◽  
Ana J. García-Sáez ◽  
...  
Keyword(s):  
Hela Cell ◽  
Quaternary Structure ◽  
Coarse Grained ◽  
Spin Labels ◽  
Cell Extracts ◽  
Protein Label ◽  
New Perspective ◽  
The Stability ◽  
Double Electron Electron Resonance

Abstract Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.

scholarly journals PREPARATION AND EVALUATION OF FAST DISSOLVING TABLETS OF PITAVASTATIN BY 32 FULL FACTORIAL DESIGN

2019 ◽  
pp. 108-114
Author(s):  
NIRMALA DASARI ◽  
VIDYAVATHI MARUVAJALA
Keyword(s):  
Drug Release ◽  
Water Absorption ◽  
Factorial Design ◽  
Disintegration Time ◽  
In Vitro Drug Release ◽  
Absorption Ratio ◽  
Conventional Film ◽  
Two Factors ◽  
The Stability

Objective: The objective of the present work was to prepare an optimized, fast dissolving tablet (FDT) of Pitavastatin to increase its dissolution by applying 32full factorial design. Methods: Nine formulations (PF1 to PF9) with all possible combinations according to 32full factorial design by selecting two factors i.e. concentration of super disintegrant, Indion414 (5-15%) (A) and sublimating agent, camphor (40-60%) (B) as independent variables at three levels of-1, 0 and 1. The effect of these two variables on three dependent parameters, water absorption ratio (Y1), disintegration time (Y2) and in vitro drug release (Y3) was studied. All the powder blends were evaluated for precompression parameters, and the tablets were prepared by direct compression method which were further evaluated for post-compression parameters. The effect of change in concentration of two selected factors on dependent parameters was studied through 3D surface response plots and polynomial equations using Design expert software version11. Optimized formula was obtained by desirability and overlay plots for which compatibility stability was assessed. Results: Precompression and post-compression parameters were satisfactorily within acceptable limits. Optimized formulation was prepared to prove the validity of the evolved mathematical model, which contained 6.75 mg of indion414(0.9) and 54 mg of camphor(0.9) with a disintegration time of 21 sec., water absorption ratio of 113 and 93% of drug release within 12 min. The compatibility between drugs and excipients was proved. The dissolution profiles of optimized formulation and commercially available conventional film-coated tablets of Pitavastatin were compared. Conclusion: The optimized formulation showed significantly (P>0.05) increased drug release compared to commercially available film-coated tablets. No changes in disintegration time, drug content and in in vitro drug release from optimized formulation on storage for 3months at 40 °C±2 °C/75% RH±5% RH were observed during stability studies which confirmed the stability of the optimized formulation.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Reverse torque values of abutment screws after dynamic loading: The effects of sealant agents and the taper of conical connections

2020 ◽  
Author(s):  
Arda Ozdiler ◽  
suleyman dayan ◽  
Burc Gencel ◽  
Gulbahar Isık-Ozkol
Keyword(s):  
Dynamic Loading ◽  
Antibacterial Agent ◽  
In Vitro Study ◽  
Control Group ◽  
Silicone Sealant ◽  
Reverse Torque ◽  
The Stability ◽  
Torque Values ◽  
Chlorhexidine Gel

This in vitro study evaluated the influence of taper angles on the internal conical connections of implant systems and of the application of chlorhexidine gel as an antibacterial agent or a polyvinyl siloxane (PVS) sealant on the reverse torque values of abutment screws after dynamic loading. The current study tested four implant systems with different taper angles (5.4°, 12°, 45°, and 60°). Specimens were divided into three groups: control (neither chlorhexidine gel filled nor silicone sealed), 2% chlorhexidine gel-filled or silicone-sealed group, and group subjected to a dynamic load of 50 N at 1 Hz for 500,000 cycles prior to reverse torque measurements. Quantitative positive correlation was observed between the taper angle degree and the percentage of tightening torque loss. However, this correlation was significant only for the 60° connection groups except in the group in which a sealant was applied ( p = 0.013 for the control group, p = 0.007 for the chlorhexidine group). Percentages of decrease in the torque values of the specimens with silicone sealant application were significantly higher compared with both the control and chlorhexidine groups ( p = 0.001, p = 0.002, p = 0.001, and p = 0.002, respectively, according to the increasing taper angles); the percentage of decrease in torque values due to chlorhexidine application was statistically insignificant when compared with the control group. The application of gel-form chlorhexidine as an antibacterial agent does not significantly affect the stability of the implant–abutment connection under dynamic loads. PVS sealants may cause screw loosening under functional loads.

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>

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

In Silico and in Vitro Evaluation of Deamidation Effects on the Stability of the Fusion Toxin DAB389IL-2

2019 ◽  
Vol 16 (4) ◽  
pp. 307-313 ◽  
Author(s):  
Nasrin Zarkar ◽  
Mohammad Ali Nasiri Khalili ◽  
Fathollah Ahmadpour ◽  
Sirus Khodadadi ◽  
Mehdi Zeinoddini
Keyword(s):  
In Silico ◽  
Catalytic Domain ◽  
Md Simulations ◽  
Special Importance ◽  
Native Form ◽  
Vitro Experiment ◽  
In Vitro Experiment ◽  
Pharmaceutical Protein ◽  
The Stability

Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation. Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated. Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment. Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

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