Biomembrane simulations of 12 lipid types using the general amber force field in a tensionless ensemble

<div><p>The AMBER family of force fields is one of the most commonly used alternatives to describe proteins and drug-like molecules in molecular dynamics simulations. However, the absence of a specific set of parameters for lipids has been limiting the widespread application of this force field in biomembrane simulations, including membrane protein simulations and drug-membrane simulations. Here, we report the systematic parameterization of 12 common lipid types consistent with the General Amber Force Field (GAFF), with charge-parameters determined with RESP at the HF/6–31G(d) level of theory, to be consistent with AMBER. The accuracy of the scheme was evaluated by comparing predicted and experimental values for structural lipid properties in MD simulations in an NPT ensemble with explicit solvent in 100:100 bilayer systems. Globally, a consistent agreement with experimental reference data on membrane structures was achieved for some lipid types when using the typical MD conditions normally employed when handling membrane proteins and drug-membrane simulations (a tensionless NPT ensemble, 310 K), without the application of any of the constraints often used in other biomembrane simulations (such as the surface tension and the total simulation box area). The present set of parameters and the universal approach used in the parameterization of all the lipid types described here, as well as the consistency with the AMBER force field family, together with the tensionless NPT ensemble used, opens the door to systematic studies combining lipid components with small drug-like molecules or membrane proteins and show the potential of GAFF in dealing with biomembranes.</p></div

Relevant Interactions of Antimicrobial Iron Chelators and Membrane Models Revealed by Nuclear Magnetic Resonance and Molecular Dynamics Simulations

The dynamics and interaction of 3-hydroxy-4-pyridinone fluorescent iron chelators, exhibiting antimicrobial properties, with biological membranes were evaluated through NMR and molecular dynamics simulations. Both NMR and MD simulation results support a strong interaction of the chelators with the lipid bilayers that seems to be strengthened for the rhodamine containing compounds, in particular for compounds that include ethyl groups and a thiourea link. For the latter type of compounds the interaction reaches the hydrophobic core of the lipid bilayer. The molecular docking and MD simulations performed for the potential interaction of the chelators with DC-SIGN receptors provide valuable information regarding the cellular uptake of these compounds since the results show that the fluorophore fragment of the molecular framework is essential for an efficient binding. Putting together our previous and present results, we put forward the hypothesis that all the studied fluorescent chelators have access to the cell, their uptake occurs through different pathways and their permeation properties correlate with a better access to the cell and its compartments and, consequently, with the chelators antimicrobial properties

Expression of iron-related genes is modulated following UPR activation.

<p>HepG2 cells were cultured in the presence of 2 mM DTT (A) or 10 mM Hcys (B) for the indicated intervals. Untreated cells were used as control. In dose-response assays, HepG2 cells were treated for 6 h with DTT (C) or Hcys (D) at concentrations ranging from 0.5 to 10 mM and from 1 to 25 mM, respectively. Control cells were incubated with vehicle. After treatments, total RNA was isolated and mRNA levels of the “iron-genes” hepcidin, ferroportin and ferritin H assessed by real-time RT-PCR. Expression of albumin, a non-iron-related gene, was evaluated as control in cells subjected to the DTT-induced ER stress. Data were normalized to <i>GAPDH</i> and are expressed as fold change over control-treated cells. Each bar displays the average+SD of three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>vs</i> control.</p

Sequences of the oligonucleotide primers used for quantitative real-time RT-PCR analysis.

<p>Sequences of the oligonucleotide primers used for quantitative real-time RT-PCR analysis.</p

DNA-binding activity of C/EBPα is modulated over the time-course of UPR activation.

<p>HepG2 cells cultured in the presence of 2 mM DTT for the indicated intervals were subjected to fEMSA (A) and ChIP (B) analyses. <i>A</i>, Nuclear protein extracts from DTT- or control-treated cells were combined with the Cy5-labeled specific probe containing the C/EBPα consensus binding site (C/EBPα SP) and electrophoresed under native conditions. For competition assays, a 50-fold molar excess of unlabeled probe (C/EBPα SC) was used (lanes 4 and 6). The specific DNA-protein complex is indicated by an arrow. Free-probe is also shown in the bottom of the gel. The fEMSA gel view displayed is representative of four independent experiments (left panel). A specific antibody against C/EBPα was added for the supershift reaction (right panel). The partial shift is highlighted in the right side of the figure. <i>B</i>, Cross-linked chromatin was immunoprecipitated with anti-C/EBPα or anti-IgG (serum control) antibodies. The recovered chromatin samples were analyzed by quantitative real-time PCR with primers flanking the C/EBPα binding site found within the −136/+9 region of human <i>HAMP</i> promoter. Results depict the enrichment relative to serum control immunoprecipitation normalized to ChIP input values and are expressed as average+SD of three independent experiments.</p

Modulation of hepcidin mRNA expression by DTT is independent of chelatable-iron.

<p>HepG2 cells were cultured in the presence of 2 mM DTT and harvested at the indicated times. For the iron chelation studies, cells were co-incubated with 20, 50 or 100 µM of DFO or L1 per culture plate. As control, cells were exposed to 50 µM of each iron chelator alone. After treatments, cDNA was synthesized from total RNA and mRNA expression levels of hepcidin were quantified by real-time RT-PCR and normalized to <i>GAPDH</i>. Data are displayed as fold change over non-treated cells and represent the average+SD of three independent experiments.</p

Involvement of clathrin-mediated endocytosis in NTBI uptake by PBMCs and HepG2 cells.

<p>(A–B) ⁵⁵Fe uptake by PBMCs and HepG2 cells in the presence of 0.45M of sucrose (A) or 80 µM of Dynasore (B) to inhibit clathrin-mediated endocytosis, after incubation with 5 µM of ⁵⁵Fe-citrate (5∶100) at 37°C (C–D) ¹²⁵I-Transferrin internalization in the presence of 0.45M of sucrose (C) or 80 µM of Dynasore (D). Each bar represents a mean value (n = 3) ± SD. Statistical significance is indicated by * symbols (*p<0.01). The inhibition of these pathways did not prevent the uptake of NTBI in both PBMCs and HepG₂ cells but transferrin internalization was decreased.</p

Kinetics of NTBI uptake in T lymphocytes and hepatocytes.

<p>NTBI uptake by human T lymphocytes (A) and HepG2 cells (B). Cells were incubated with different concentrations of ⁵⁵Fe-citrate (1 µM, 5 µM, 10 µM, 100 µM, 200 µM and 500 µM) at 37°C and intracellular iron quantified at various time points (0, 15, 30, 60 and 120 min) (n = 3). The values obtained during the first 30 min of incubation, when the transport system is not saturated, were used to calculate the rate of uptake for each concentration. CD3⁺ cells reach saturation at 200 µM of Fe-citrate and present a maximum rate of 0.4 nmol/min/10⁶ cells, as opposite to HepG2 cells, which do not saturate even at 500 µM and present a faster rate of uptake (21 nmol/min/10⁶ cells).</p

Involvement of NTBI transporters in Ferric citrate uptake by T lymphocytes.

<p>mRNA levels (A; qRT-PCR) and corresponding NTBI uptake (B) by CD4⁺ and CD8⁺ T lymphocytes following nucleofection with siRNAs specific for ZIP14, DMT1-IRE and DMT1-nIRE, with scrambled siRNAs (siNeg) or with no DNA as controls. Each column represents the mean value (n = 3) ± SD. Statistical significance between samples (grey columns) and controls (white columns) is indicated by * symbols (*p<0.01). No differences in Fe∶citrate uptake were observed after silencing DMT1 or ZIP14.</p

Similar patterns of NTBI uptake by T lymphocytes and hepatocytes.

<p>(A) NTBI uptake by human T-lymphocytes. CD4⁺ and CD8⁺ human T-lymphocytes were incubated with 5 µM of ⁵⁵Fe-citrate (5∶100) at 37°C and 4°C and intracellular iron quantified at each time-point. Each point = average (n≥3) ±1SD. (B) NTBI uptake by HepG2 cells. HepG2 cells were incubated with 5 µM of ⁵⁵Fe-citrate (5∶100) for up to 24 hours, at 37°C. Cell-associated ⁵⁵Fe levels at each time point were measured. Each point is a mean value (n = 6) ± SD. Both T-lymphocytes and HepG2 cells are able to accumulate NTBI presenting a high rate of uptake during the first 30 minutes of incubation (C–D) Specificity of NTBI uptake. CD3⁺ cells were incubated with 5 µM of ⁵⁵Fe-citrate (5∶100) for up to 90 min, at 37°C (C) or 4°C (D), and at each time point washed either with PBS (with or without pronase) or incubated for 15 min with serum-free RPMI with trypsin. Cell-associated ⁵⁵Fe levels at each time point were measured. Each point is a mean value (n = 3) ± SD. The similar results obtained at 37°C together with the differences at 4°C suggest that most of the measured iron is intracellular. Statistical significance between samples at 37°C and controls at 4°C is indicated by * symbols (*p<0.01).</p