scholarly journals Water channel pore size determines exclusion properties but not solute selectivity

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Philip Kitchen ◽  
Mootaz M. Salman ◽  
Simone U. Pickel ◽  
Jordan Jennings ◽  
Susanna Törnroth-Horsefield ◽  
...  
Keyword(s):  
Pore Size ◽  
In Silico ◽  
Water Channel ◽  
Water Channels ◽  
Selectivity Filter ◽  
Single Amino Acid ◽  
Cross Sectional ◽  
Solute Permeability ◽  
Channel Proteins

AbstractAquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels also facilitates transmembrane diffusion of small, polar solutes. A constriction within the pore, the aromatic/arginine (ar/R) selectivity filter, is thought to control solute permeability: previous studies on single representative water channel proteins suggest narrow channels conduct water, whilst wider channels permit passage of solutes. To assess this model of selectivity, we used mutagenesis, permeability measurements and in silico comparisons of water-specific as well as glycerol-permeable human AQPs. Our studies show that single amino acid substitutions in the selectivity filters of AQP1, AQP4 and AQP3 differentially affect glycerol and urea permeability in an AQP-specific manner. Comparison between in silico-calculated channel cross-sectional areas and in vitro permeability measurements suggests that selectivity filter cross-sectional area predicts urea but not glycerol permeability. Our data show that substrate discrimination in water channels depends on a complex interplay between the solute, pore size, and polarity, and that using single water channel proteins as representative models has led to an underestimation of this complexity.

Conformational dynamics and interfacial interactions of peptide-appended pillar[5]arene water channels in biomimetic membranes

2019 ◽  
Vol 21 (41) ◽  
pp. 22711-22721 ◽  
Author(s):  
Yong Liu ◽  
Harish Vashisth
Keyword(s):  
Water Purification ◽  
Conformational Dynamics ◽  
Water Channel ◽  
Water Channels ◽  
Interfacial Interactions ◽  
Biomimetic Membranes ◽  
Channel Proteins ◽  
Significant Potential ◽  
Biological Water ◽  
Artificial Water

Peptide appended pillar[5]arene (PAP) is an artificial water channel resembling biological water channel proteins, which has shown a significant potential for designing bioinspired water purification systems.

In-silico structural, functional and immunogenic characterization of Taenia solium TS14 protein

2017 ◽  
Author(s):  
Chitra Joshi ◽  
Siddharth Gautam
Keyword(s):  
Amino Acid ◽  
In Silico ◽  
Mutational Analysis ◽  
Solvent Accessibility ◽  
Taenia Solium ◽  
Secretory Protein ◽  
Single Amino Acid

TS14, a Cysticercosis cellulosae derived protein, has been exploited for immunodiagnosis of cysticercosis in humans and pigs. However, the information on structure, function, stability and immunogenicity of TS14 derived from different isolates is primarily lacking. The present study deals with in-silico characterization of six TS14 isolates. High thermostability and an isoelectric point of 9.41 were recorded. Based on N-terminal amino acid residues, high resistance to intracellular proteases with extended in-vivo and in-vitro half-lives was predicted. TS14 is foreseen as a secretory protein with a signal peptide and an extracellular localization. Structural analysis of TS14 exhibited the dominance of helices in the secondary structure (92% coverage) with majority of residues showing high and medium solvent accessibility. High lysine content and presence of multiple nucleotide binding sites in TS14 suggests interaction with RNA/DNA and a role in their metabolism. Immunogenic profiling predicted presence of four distinct B-cell epitopes. Mutational analysis based on the single amino acid substitutions among six TS14 isolates demonstrated minor variations in structural stability; however, all the substitutions were well tolerated. Moreover, all the isolates revealed almost identical immunogenic profile with an equivocal potential to elicit the antibody-mediated immune response.

scholarly journals Lessons from a Single Amino Acid Substitution: Anticancer and Antibacterial Properties of Two Phospholipase A2-Derived Peptides

2021 ◽  
Vol 44 (1) ◽  
pp. 46-62
Author(s):  
José R. Almeida ◽  
Bruno Mendes ◽  
Marcelo Lancellotti ◽  
Gilberto C. Franchi ◽  
Óscar Passos ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phospholipase A2 ◽  
Snake Venom ◽  
Amino Acid Substitution ◽  
In Silico ◽  
Fine Tuning ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Significant Activity

The membrane-active nature of phospholipase A2-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA2 toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A2 isoforms, is again demonstrated as a valuable source of therapeutic peptides.

Ion/Water Channels for Embryo Implantation Barrier

Physiology ◽  
2014 ◽  
Vol 29 (3) ◽  
pp. 186-195 ◽  
Author(s):  
Xin-Mei Liu ◽  
Dan Zhang ◽  
Ting-Ting Wang ◽  
Jian-Zhong Sheng ◽  
He-Feng Huang
Keyword(s):  
Stromal Cells ◽  
Action Potentials ◽  
Steroid Hormone ◽  
Embryo Implantation ◽  
Water Channel ◽  
Water Channels ◽  
Resting Membrane Potential ◽  
Luminal Epithelium ◽  
Channel Proteins ◽  
Harmful Substances

Successful implantation involves three distinct processes, namely the embryo apposition, attachment, and penetration through the luminal epithelium of the endometrium to establish a vascular link to the mother. After penetration, stromal cells underlying the epithelium differentiate and surround the embryo to form the embryo implantation barrier, which blocks the passage of harmful substances to the embryo. Many ion/water channel proteins were found to be involved in the process of embryo implantation. First, ion/water channel proteins play their classical role in establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane. Second, most of ion/water channel proteins are regulated by steroid hormone (estrogen or progesterone), which may have important implications to the embryo implantation. Last but not least, these proteins do not limit themselves as pure channels but also function as an initiator of a series of consequences once activated by their ligand/stimulator. Herein, we discuss these new insights in recent years about the contribution of ion/water channels to the embryo implantation barrier construction during early pregnancy.

Fate of antidiuretic hormone water channel proteins after retrieval from apical membrane

1993 ◽  
Vol 265 (3) ◽  
pp. C822-C833 ◽  
Author(s):  
M. L. Zeidel ◽  
T. G. Hammond ◽  
J. B. Wade ◽  
J. Tucker ◽  
H. W. Harris
Keyword(s):  
Membrane Proteins ◽  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Water Channel ◽  
Fluid Phase ◽  
Water Channels ◽  
Endocytic Pathway ◽  
Electron Microscopic ◽  
Channel Proteins ◽  
Fluorescence Measurements

In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.

scholarly journals In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant

2020 ◽  
Vol 21 (16) ◽  
pp. 5857
Author(s):  
Michal Cohen ◽  
Emanuele Pignatti ◽  
Monica Dines ◽  
Adi Mory ◽  
Nina Ekhilevitch ◽  
...  
Keyword(s):  
In Silico ◽  
Structure Prediction ◽  
De Novo ◽  
Pathogenic Variant ◽  
Single Amino Acid ◽  
Evolutionary Information ◽  
Evolutionary Analysis ◽  
Cyp21a2 Gene ◽  
Salt Wasting

Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patient, 46 XX newborn, was diagnosed with classical salt wasting CAH in the neonatal period after initially presenting with ambiguous genitalia. Multiplex ligation-dependent probe analysis demonstrated a full deletion of the paternal CYP21A2 gene, and Sanger sequencing revealed a novel de novo CYP21A2 variant c.694–696del (E232del) in the other allele. This variant resulted in the deletion of a non-conserved single amino acid, and its functional relevance was initially undetermined. We used both in silico and in vitro methods to determine the mechanistic significance of this mutation. Computational analysis relied on the solved structure of the protein (Protein-data-bank ID 4Y8W), structure prediction of the mutated protein, evolutionary analysis, and manual inspection. We predicted impaired stability and functionality of the protein due to a rotatory disposition of amino acids in positions downstream of the deletion. In vitro biochemical evaluation of enzymatic activity supported these predictions, demonstrating reduced protein levels to 22% compared to the wild-type form and decreased hydroxylase activity to 1–4%. This case demonstrates the potential of combining in-silico analysis based on evolutionary information and structure prediction with biochemical studies. This approach can be used to investigate other genetic variants to understand their potential effects.

scholarly journals In silico-guided sequence modifications of K-ras epitopes improve immunological outcome against G12V and G13D mutant KRAS antigens

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5056 ◽  
Author(s):  
Allan Wee Ren Ng ◽  
Pei Jun Tan ◽  
Winfrey Pui Yee Hoo ◽  
Dek Shen Liew ◽  
Michelle Yee Mun Teo ◽  
...  
Keyword(s):  
B Cell ◽  
In Silico ◽  
Secondary Structure Prediction ◽  
Cell Epitope ◽  
Peptide Sequence ◽  
Single Amino Acid ◽  
Peptide Vaccines ◽  
Exon 2 ◽  
B Cell Epitopes

Background Somatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions. Methods In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA. Results In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls. Discussion In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.

An in vitro Comparison of Two Replacement Techniques Utilizing Fascia Lata after Cranial Cruciate Ligament Transection in the Dog

1993 ◽  
Vol 06 (02) ◽  
pp. 85-92 ◽  
Author(s):  
G. L. Coetzee
Keyword(s):  
Femoral Condyle ◽  
Cruciate Ligament ◽  
Maximum Load ◽  
Fascia Lata ◽  
Patellar Ligament ◽  
Cross Sectional ◽  
Cranial Cruciate Ligament ◽  
Replacement Technique ◽  
Over The Top

SummaryThe immediate postoperative biomechanical properties of an “underand-over” cranial cruciate ligament (CCL) replacement technique consisting of fascia lata and the lateral onethird of the patellar ligament, were compared with that of a modified intra- and extracapsular “under-and-over-the-top” (UOTT) method. The right CCL in twelve adult dogs was dissected out and replaced with an autograft. The contralateral, intact CCL served as the control. In group A, the graft was secured to the lateral femoral condyle with a spiked washer and screw. In group B the intracapsular graft was secured to the lateral femoro-fabellar ligament, and the remainder to the patellar tendon. Both CCL replacement techniques exhibited a 2.0 ± 0.5 mm anterior drawer immediately after the operation. After skeletonization of the stifles, the length and cross-sectional area of the intact CCL and CCL substitutes were determined. Each bone-ligament unit was tested in linear tension to failure at a fixed distraction rate of 15 mm/s with the stifle in 120° flexion. Data was processed to obtain the corresponding material parameters (modulus, stress and strain in the linear loading region, and energy absorption to maximum load).The immediate postoperative structural and material properties of the “under-and-over” cranial cruciate ligament replacement technique with autogenous fascia lata, were compared to that of a modified intra- and extracapsular “under-and-over-the-top” (UOTT) method. The combined UOT T technique was slightly stronger (6%), but allowed 2.8 ± 0.9 mm more cranial tibial displacement at maximum linear force.

IN VITRO/IN SILICO CHARACTERIZATION OF ACTIVE AND PASSIVE STRESSES IN CARDIAC MUSCLE

2012 ◽  
Vol 10 (2) ◽  
pp. 171-188 ◽  
Author(s):  
Markus Boel ◽  
Oscar J. Abilez ◽  
Ahmed N Assar ◽  
Christopher K. Zarins ◽  
Ellen Kuhl
Keyword(s):  
Cardiac Muscle ◽  
In Silico ◽  
In Silico Characterization

Role of 4-O Galloylchlorogenic Acid in Lung Cancer- An Insilico Approach

2017 ◽  
Vol 9 (5) ◽  
Author(s):  
Jaynthy C. ◽  
N. Premjanu ◽  
Abhinav Srivastava
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
In Silico ◽  
Computational Study ◽  
Regulation Mechanism ◽  
Down Regulation ◽  
Fruit Extract ◽  
In Vitro Techniques ◽  
Discovery Studio

Cancer is a major disease with millions of patients diagnosed each year with high mortality around the world. Various studies are still going on to study the further mechanisms and pathways of the cancer cell proliferation. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. In cancer development increased core fucosylation by FUT8 play an important role in cell proliferation. Down regulation of FUT8 expression may help cure lung cancer. Therefore the computational study based on the down regulation mechanism of FUT8 was mechanised. Sapota fruit extract, containing 4-Ogalloylchlorogenic acid was used as the inhibitor against FUT-8 as target and docking was performed using in-silico tool, Accelrys Discovery Studio. There were several conformations of the docked result, and conformation 1 showed 80% dock score between the ligand and the target. Further the amino acids of the inhibitor involved in docking were studied using another tool, Ligplot. Thus, in-silico analysis based on drug designing parameters shows that the fruit extract can be studied further using in-vitro techniques to know its pharmacokinetics.

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