Standard binding free energy and membrane desorption mechanism for a phospholipase C

Peripheral membrane proteins (PMPs) bind temporarily to cellular membranes and play important roles in signalling, lipid metabolism and membrane trafficking. Obtaining accurate membrane-PMP affinities using experimental techniques is more challenging than for protein-ligand affinities in aqueous solution. At the theoretical level, calculation of standard protein-membrane binding free energy using molecular dynamics simulations remains a daunting challenge owing to the size of the biological objects at play, the slow lipid diffusion and the large variation in configurational entropy that accompanies the binding process. To overcome these challenges, we used a computational framework relying on a series of potential-of-mean-force (PMF) calculations including a set of geometrical restraints on collective variables. This methodology allowed us to determine the standard binding free energy of a PMP to a phospholipid bilayer using an all-atom force field. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) was chosen due to its importance as a virulence factor and owing to the host of experimental affinity data available. We computed a standard binding free energy of -8.2±1.4 kcal/mol in reasonable agreement with the reported experimental values (-6.6±0.2 kcal/mol). In light of the 2.3-μs separation PMF calculation, we investigated the mechanism whereby BtPI-PLC disengages from interactions with the lipid bilayer during separation. We describe how a short amphipathic helix engages in transitory interactions to ease the passage of its hydrophobes through the interfacial region upon desorption from the bilayer.

The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

The activity of the intrinsically water-soluble enzyme ADAMTS13 correlates with the membrane state when bound to a phospholipid bilayer

Membrane-associated enzymes have been found to behave differently qualitatively and quantitatively in terms of activity. These findings were highly debated in the 1970s and many general correlations and reaction specific models have been proposed, reviewed, and discarded. However, new biological applications brought up the need for clarification and elucidation. To address literature shortcomings, we chose the intrinsically water-soluble enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) and large unilamellar vesicles with a relative broad phase transition. We here present activity measurements of ADAMTS13 in the freely dissolved state and the membrane associated state for phosphocholine lipids with different acyl-chain lengths (13:0, 14:0 and 15:0) and thus main phase transition temperatures. While the freely dissolved enzyme shows a simple Arrhenius behavior, the activity of membrane associated ADAMTS13 in addition shows a peak. This peak temperature correlates with the main phase transition temperature of the used lipids. These findings support an alternative theory of catalysis. This theory predicts a correlation of the membrane associated activity and the heat capacity, as both are susceptibilities of the same surface Gibb’s free energy, since the enzyme is attached to the membrane.

Molecular Characterization of CNTS Mutations in Tunisian Patients with Ocular Cystinosis

Background: Ocular cystinosis is a rare autosomal recessive disorder characterized by intralysosomal cystine accumulation in renal, ophthalmic (cornea, conjunctiva), and other organ abnormalities. Patients with ocular cystinosis are mostly asymptomatic and typically experience mild photophobia due to cystine crystals in the cornea observed accidently during a routine ocular examination. The ocular cystinosis is associated with different mutations in CTNS gene. Cysteamine therapy mostly corrects the organ abnormalities. Methods: This study was performed in collaboration with the department of ophthalmology of Farhat Hached Hospital. The Optical Coherence Tomography (OCT) of the cornea and retinal photography were used to search cystine crystals within the corneas and conjunctiva in eight Tunisian patients. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the entire CTNS gene. Results: The studied patients were found to have cystine crystal limited anterior corneal stroma and the conjunctiva associated with retinal crystals accumulation. CTNS gene sequencing disclosed 7 mutations: three missense mutations (G308R, p.Q88K, and p.S139Y); one duplication (C.829dup), one framshift mutation (p.G258f), one splice site mutation (c.681+7delC) and a large deletion (20327-bp deletion). Crystallographic structure analysis suggests that the novel mutation p.S139Y is buried in a first transmembrane helix closed to the lipid bilayer polar region, introducing a difference in hydrophobicity which could affect the hydrophobic interactions with the membrane lipids. The second novel mutation p.Q88K which is located in the lysosomal lumen close to the lipid membrane polar head region, introduced a basic amino acid in a region which tolerate only uncharged residue. The third missense mutation introduces a positive change in nonpolar tail region of the phospholipid bilayer membrane affecting the folding and stability of the protein in the lipid bilayer. Conclusion: Our data demonstrate that impaired transport of cystine out of lysosomes is the most common, which is obviously associated with the mutations of transmembrane domains of cystinosine resulting from a total loss of its activity.

The Impact of an Anchoring Layer on the Formation of Tethered Bilayer Lipid Membranes on Silver Substrates

Tethered bilayer lipid membranes (tBLMs) have been known as stable and versatile experimental platforms for protein–membrane interaction studies. In this work, the assembly of functional tBLMs on silver substrates and the effect of the molecular chain-length of backfiller molecules on their properties were investigated. The following backfillers 3-mercapto-1-propanol (3M1P), 4-mercapto-1-butanol (4M1B), 6-mercapto-1-hexanol (6M1H), and 9-mercapto-1-nonanol (9M1N) mixed with the molecular anchor WC14 (20-tetradecyloxy-3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol) were used to form self-assembled monolayers (SAMs) on silver, which influenced a fusion of multilamellar vesicles and the formation of tBLMs. Spectroscopic analysis by SERS and RAIRS has shown that by using different-length backfiller molecules, it is possible to control WC14 anchor molecules orientation on the surface. An introduction of increasingly longer surface backfillers in the mixed SAM may be related to the increasing SAMs molecular order and more vertical orientation of WC14 at both the hydrophilic ethylenoxide segment and the hydrophobic lipid bilayer anchoring alkane chains. Since no clustering of WC14 alkane chains, which is deleterious for tBLM integrity, was observed on dry samples, the suitability of mixed-component SAMs for subsequent tBLM formation was further interrogated by electrochemical impedance spectroscopy (EIS). EIS showed the arrangement of well-insulating tBLMs if 3M1P was used as a backfiller. An increase in the length of the backfiller led to increased defectiveness of tBLMs. Despite variable defectiveness, all tBLMs responded to the pore-forming cholesterol-dependent cytolysin, vaginolysin in a manner consistent with the functional reconstitution of the toxin into phospholipid bilayer. This experiment demonstrates the biological relevance of tBLMs assembled on silver surfaces and indicates their utility as biosensing elements for the detection of pore-forming toxins in liquid samples.

Blood Nanoparticles – Influence on Extracellular Vesicle Isolation and Characterization

Blood is a rich source of disease biomarkers, which include extracellular vesicles (EVs). EVs are nanometer-to micrometer-sized spherical particles that are enclosed by a phospholipid bilayer and are secreted by most cell types. EVs reflect the physiological cell of origin in terms of their molecular composition and biophysical characteristics, and they accumulate in blood even when released from remote organs or tissues, while protecting their cargo from degradation. The molecular components (e.g., proteins, miRNAs) and biophysical characteristics (e.g., size, concentration) of blood EVs have been studied as biomarkers of cancers and neurodegenerative, autoimmune, and cardiovascular diseases. However, most biomarker studies do not address the problem of contaminants in EV isolates from blood plasma, and how these might affect downstream EV analysis. Indeed, nonphysiological EVs, protein aggregates, lipoproteins and viruses share many molecular and/or biophysical characteristics with EVs, and can therefore co-isolate with EVs from blood plasma. Consequently, isolation and downstream analysis of EVs from blood plasma remain a unique challenge, with important impacts on the outcomes of biomarker studies. To help improve rigor, reproducibility, and reliability of EV biomarker studies, we describe here the major contaminants of EV isolates from blood plasma, and we report on how different EV isolation methods affect their levels, and how contaminants that remain can affect the interpretation of downstream EV analysis.

Latest Trend of Milk Derived Exosomes: Cargos, Functions, and Applications

Exosomes are nanosized phospholipid bilayer vesicles released to the extracellular environment. Exosomes from various tissues or cells are being studied and there has been a growing interest in milk exosomes research due to their emerging role as messengers between cells and the fact that it can be produced in large quantities with rich source of milk. Milk derived exosomes (MDEs) contain lipids, microRNAs, proteins, mRNAs as well as DNA. Studies of exosome cargo have been conducted widely in many research areas, especially exosomal miRNAs. In this paper, we reviewed the current knowledge in isolation and identification, cargos, functions mainly in intestinal tract and immunity system of MDEs. Its application as drug carriers and diseases biomarker are also discussed. Furthermore, we also consider critical challenges of MDEs application and provide possible directions for future research.

Experimental Evaluation of Food-Grade Semi-Refined Carrageenan Toxicity

The safety of food additives E407 and E407a has raised concerns in the scientific community. Thus, this study aims to assess the local and systemic toxic effects of the common food additive E407a in rats orally exposed to it for two weeks. Complex evaluations of the effects of semi-refined carrageenan (E407a) on rats upon oral exposure were performed. Local effects of E407a on the intestine were analyzed using routine histological stains and CD68 immunostaining. Furthermore, circulating levels of inflammatory markers were assessed. A fluorescent probe O1O (2- (2′-OH-phenyl)-5-phenyl-1,3-oxazole) was used for evaluating the state of leukocyte cell membranes. Cell death modes of leukocytes were analyzed by flow cytometry using Annexin V and 7-aminoactinomycin D staining. Oral administration of the common food additive E407a was found to be associated with altered small and large intestinal morphology, infiltration of the lamina propria in the small intestine with macrophages (CD68+ cells), high systemic levels of inflammation markers, and changes in the lipid order of the phospholipid bilayer in the cell membranes of leukocytes, alongside the activation of their apoptosis. Our findings suggest that oral exposure to E407a through rats results in the development of intestinal inflammation.