Cooperative cation co-transport by supramolecular self-associating amphiphiles

We report investigations into the cation transport ability of a series of antimicrobial supramolecular, self-associating amphiphiles (SSAs). The SSAs proved to be challenging to study using standard, literature assays and we therefore describe a novel methodology to examine cation transport via a chloride co-transport assay. One SSA in this series was observed to function as a K+ and Na+ transporter with promising deliverability properties. The results shed light on a potential mechanism of microbial toxicity and inform the design of future targets.

Supramolecular methods: the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) transport assay

The vesicular anion transport activity assay, which uses 8-hydroxypyrene-1,3,6-trisulfonic acid to monitor the internal pH of the vesicles (the HPTS assay), is a widely used technique for analysing the activity of anionophore facilitated transport across a phospholipid membrane. This methods paper describes the stepwise technique to conduct this transport assay, detailing both the perks and pitfalls of using this method to determine the activity of an anionophore and the transport mechanism.

Molecular dynamics and theratyping in airway and gut organoids reveal R352Q-CFTR conductance defect

A significant challenge to making targeted CFTR modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q — with potential role contributing to intrapore chloride ion permeation — in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using ion transport assay and forskolin-induced swelling (FIS) assay respectively. Two CFTR potentiators (VX-770, GLPG1837) and a corrector (VX-809) were tested. Data from R352Q-CFTR were compared to that of participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function which was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics (MD) simulations of R352Q-CFTR were carried out which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico MD simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Localization, proteomics, and metabolite profiling reveal a putative vesicular transporter for UDP-glucose

Vesicular neurotransmitter transporters (VNTs) mediate the selective uptake and enrichment of small molecule neurotransmitters into synaptic vesicles (SVs) and are therefore a major determinant of the synaptic output of specific neurons. To identify novel VNTs expressed on SVs (thus identifying new neurotransmitters and/or neuromodulators), we conducted localization profiling of 361 solute carrier (SLC) transporters tagging with a fluorescent protein in neurons, which revealed 40 possible candidates through comparison with a known SV marker. We parallelly performed proteomics analysis of immunoisolated SVs and identified 7 transporters in overlap. Ultrastructural analysis confirmed one of the transporters, SLC35D3, localized to SVs. Finally, by combining metabolite profiling with a radiolabeled substrate transport assay, we identified UDP-glucose as the principal substrate for SLC35D3. These results provide new insights into the functional role of SLC transporters in neurotransmission and improve our understanding of the molecular diversity of chemical transmitters.

Characterization and Caco-2 Cell Transport Assay of Chito-Oligosaccharides Nano-Liposomes Based on Layer-by-Layer Coated

Chito-oligosaccharides (COSs) were encapsulated by the film-ultrasonic method into three nano-liposomes, which were uncoated liposomes (COSs-Lip), chitosan-coated liposomes (CH-COSs-Lip), and sodium alginate (SA)/chitosan (CH)-coated liposomes (SA/CH-COSs-Lip). The physicochemical and structural properties, as well as the stability and digestive characteristics, of all three nano-liposomes were assessed in the current study. Thereafter, the characteristics of intestinal absorption and transport of nano-liposomes were investigated by the Caco-2 cell monolayer. All nano-liposomes showed a smaller-sized distribution with a higher encapsulation efficiency. The ζ-potential, Z-average diameter (Dz), and polydispersity index (PDI) demonstrated that the stability of the SA/CH-COSs-Lip had much better stability than COSs-Lip and CH-COSs-Lip. In addition, the transport of the nano-liposomes via the Caco-2 cell monolayer indicated a higher transmembrane transport capacity. In summary, the chitosan and sodium alginate could serve as potential delivery systems for COSs to fortify functional foods and medicines.

Depletion of Branched-Chain Ketoacid Dehydrogenase Kinase (BDK) Upregulates Insulin-Stimulated Glucose Uptake in Muscle Cells

Objectives Plasma levels of branched-chain amino acids (BCAAs) and their metabolites, branched-chain ketoacids (BCKAs) are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus (T2DM). BCAA catabolic enzymes are downregulated in diabetes and obesity. We previously showed that leucine and KIC suppressed insulin-stimulated glucose uptake in L6 myotubes. We have also shown that knocking down branched-chain ketoacid dehydrogenase (BCKD), an enzyme that decarboxylates BCKAs, suppressed insulin-stimulated glucose uptake. The objective of this study is to analyze how stimulating BCAA catabolic flux, by depleting branched-chain ketoacid dehydrogenase kinase (BDK), a negative regulator of BCKD, affects insulin sensitivity. We hypothesize that upregulating BCAA catabolism will increase insulin-stimulated glucose transport and attenuate insulin resistance. Methods L6 myoblasts were cultured in differentiation media for 4 days. On day 4 of differentiation, cells were transfected with control (SCR) or branched-chain ketoacid dehydrogenase kinase (BDK) siRNA oligonucleotides. Forty-eight hours later, myotubes were starved of serum- and amino acids for 3 hours then supplemented with or without KIC (200 mM) for 30 minutes. After, cells were incubated with or without insulin (100 nM) for 20 minutes. They were then harvested for immunoblotting or used for glucose transport assay. Results There was a 32% increase in insulin-stimulated glucose uptake with BDK depletion. KIC suppressed insulin-stimulated glucose uptake by 25% in control (SCR) cells; this suppression was attenuated in cells depleted of BDK. BDK depletion also reduced KIC-induced IRS-1Ser612 phosphorylation by 64% but had no effect on AktSer473 phosphorylation. Conclusions BDK depletion increased insulin-stimulated glucose transport, and attenuated KIC-induced suppression of insulin-stimulated glucose uptake, suggesting that increasing BCKD activity can be a therapeutic strategy against insulin resistance. Funding Sources Natural Science and Research Council (NSERC)

Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

In mammalian cells, the L-type amino acid transporters (LATs) LAT1 (SLC7A5) and LAT2 (SLC7A8) form heterodimeric amino acid transporters (HATs) with the ancillary protein 4F2hc and are involved in the cellular uptake of specific amino acids. The HAT 4F2hc-LAT1 is found upregulated in various cancer cell types, while 4F2hc-LAT2 is a transporter for non-cancer cells. Preclinical studies have highlighted that 4F2hc-LAT1 plays an important role in tumor progression representing a valid anticancer target. Consequently, current research is focusing on the development of potent and specific human 4F2hc-LAT1 inhibitors. On the other hand, 4F2hc-LAT2 is emerging as target of other diseases, thus also gaining clinical interest. To determine affinity and specificity of substrates and inhibitors for 4F2hc-LAT1 or 4F2hc-LAT2, robust transport cell assays are indispensable. We have optimized and validated a transport assay using cells of the methylotrophic yeast Pichia pastoris stably overexpressing the human HATs 4F2hc-LAT1 or -LAT2, and the LATs LAT1 or LAT2 alone. The radioligand [3H]L-leucine was used as reporter and the substrates L-leucine, triiodothyronine (T3) and thyroxine (T4) as well as the inhibitors BCH and JPH203 (KYT-0353) for assay validation. Obtained half-maximal inhibitory concentrations also provided new insights, e.g., into the LAT specificity of the potent inhibitor JPH203 and on the potency of the thyroid hormones T3 and T4 to inhibit transport through human 4F2hc-LAT2. The LAT1 and LAT2 assays are of particular interest to determine possible implications and influences of 4F2hc in ligand binding and transport. In summary, the presented assays are valuable for characterization of ligands, e.g., towards 4F2hc-LAT1 specificity, and can also be applied for compound screening. Finally, our established approach and assay would also be applicable to other HATs and LATs of interest.