Counter-directed leucine gradient promotes amino acid transfer across the human placenta.

The developing fetus is highly vulnerable to imbalances in the supply of essential amino acids (AA). Transplacental AA transfer depends on complex interactions between accumulative transporters, exchangers and facilitators, which maintain both intra-extracellular and materno-fetal substrate gradients. We determined physiological AA gradients between maternal and fetal blood and assessed their importance by studying maternal-fetal leucine transfer in human trophoblasts. Maternal-venous and corresponding fetal-arterial/fetal-venous sera were collected from 22 healthy patients at partum. The acquisition of the full AA spectra in serum was performed by ion exchange chromatography. Physiological materno-fetal AA levels were evaluated using paired two-way ANOVA with Tukey's correction. AA concentrations and gradients were tested for associations with anthropometric data by Spearman correlation analysis. Functional effects of a physiological leucine gradient versus equimolar concentrations were tested in BeWo cells using L-[3H]-leucine in conventional and Transwell-based uptake and transfer experiments. The LAT1/SLC7A5-specific inhibitor JPH203 was used to evaluate LAT1-transporter-mediated leucine transport. Maternal AA concentrations correlated with preconceptional and maternal weights at partum. Interestingly, low materno-fetal AA gradients were associated with maternal weight, BMI and gestational weight gain. Leucine uptake was promoted by increased extracellular substrate concentrations. Materno-fetal leucine transfer was significantly increased against a 137µM leucine gradient demonstrating that transplacental leucine transport is stimulated by a counter-directed gradient. Moreover, leucine transfer was inhibited by 10µM JPH203 confirming that Leu transport across the trophoblast monolayer is LAT1-dependent. This study demonstrates a currently underestimated effect of transplacental AA gradients on efficient leucine transfer which could severely affect fetal development.

Small molecule inhibitors provide insights into the relevance of LAT1 and LAT2 in materno-foetal amino acid transport.

The placenta supplies the foetus with critical nutrients such as essential amino acids (AA, eg leucine) for development and growth. It also represents a cellular barrier which is formed by a polarized, differentiated syncytiotrophoblast (STB) monolayer. Active Na+ -independent leucine transport across the placenta is mainly attributed to the System L transporters LAT1/SLC7A5 and LAT2/SLC7A8. This study explored the influence of trophoblast differentiation on the activity of LAT1/LAT2 and the relevance of LAT1/LAT2 in leucine uptake and transfer in trophoblasts by applying specific small molecule inhibitors (JPH203/JG336/JX009). L-leucine uptake (total dose = 167 µmol/L) was sensitive to LAT1-specific inhibition by JPH203 (EC50  = 2.55 µmol/L). The inhibition efficiency of JPH203 was increased by an additional methoxy group in the JPH203-derivate JG336 (EC50  = 1.99 µmol/L). Interestingly, JX009 showed efficient System L inhibition (EC50  = 2.35 µmol/L) and was the most potent inhibitor of leucine uptake in trophoblasts. The application of JPH203 and JX009 in Transwell® -based leucine transfer revealed LAT1 as the major accumulative transporter at the apical membrane, but other System L transporters such as LAT2 as rate-limiting for leucine efflux across the basal membrane. Therefore, differential specificity of the applied inhibitors allowed for estimation of the contribution of LAT1 and LAT2 in materno-foetal AA transfer and their potential impact in pregnancy diseases associated with impaired foetal growth.

Knotting and unknotting of a protein in single molecule experiments.

Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.

The complex folding network of single calmodulin molecules.

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.

Ligand-dependent equilibrium fluctuations of single calmodulin molecules.

Single-molecule force spectroscopy allows superb mechanical control of protein conformation. We used a custom-built low-drift atomic force microscope to observe mechanically induced conformational equilibrium fluctuations of single molecules of the eukaryotic calcium-dependent signal transducer calmodulin (CaM). From this data, the ligand dependence of the full energy landscape can be reconstructed. We find that calcium ions affect the folding kinetics of the individual CaM domains, whereas target peptides stabilize the already folded structure. Single-molecule data of full length CaM reveal that a wasp venom peptide binds noncooperatively to CaM with 2:1 stoichiometry, whereas a target enzyme peptide binds cooperatively with 1:1 stoichiometry. If mechanical load is applied directly to the target peptide, real-time binding/unbinding transitions can be observed.