Safety of Dietary Guanidinoacetic Acid: A Villain of a Good Guy?

Guanidinoacetic acid (GAA) is a natural amino acid derivative that is well-recognized for its central role in the biosynthesis of creatine, an essential compound involved in cellular energy metabolism. GAA (also known as glycocyamine or betacyamine) has been investigated as an energy-boosting dietary supplement in humans for more than 70 years. GAA is suggested to effectively increase low levels of tissue creatine and improve clinical features of cardiometabolic and neurological diseases, with GAA often outcompeting traditional bioenergetics agents in maintaining ATP status during stress. This perhaps happens due to a favorable delivery of GAA through specific membrane transporters (such as SLC6A6 and SLC6A13), previously dismissed as un-targetable carriers by other therapeutics, including creatine. The promising effects of dietary GAA might be countered by side-effects and possible toxicity. Animal studies reported neurotoxic and pro-oxidant effects of GAA accumulation, with exogenous GAA also appearing to increase methylation demand and circulating homocysteine, implying a possible metabolic burden of GAA intervention. This mini-review summarizes GAA toxicity evidence in human nutrition and outlines functional GAA safety through benefit-risk assessment and multi-criteria decision analysis.

PMRT1, a Plasmodium specific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures – the food vacuole, the apicoplast, and the parasite plasma membrane – and showed essentiality of four out of the six membrane transporters during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis. Heterologous expression of wild-type and mutation constructs in Xenopus laevis oocytes indicated ion transport upon membrane hyperpolarization and a functional role of negatively charged amino acids protruding into the parasitophorous vacuole lumen. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development and provide the first functional characterization of PfPMRT1, an essential parasite membrane transporter.

Cyanidin 3-glucoside Is a Selective Inhibitor of Hepatic Bilirubin Uptake

Background & Aims: One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, as there is no link to the protein/gene entities that carry it out, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology.Methods: We treated isolated, perfused rat livers with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a biofluorometric assay. We tested the ability of nine molecules known to be substrates or inhibitors of sinusoidal membrane transporters to inhibit the hepatic uptake of bilirubin.Results: We found that cyanidin 3-glucoside and malvidin 3-glucoside are the only molecules that inhibit bilirubin uptake. These dietary anthocyanins resemble bromosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cyanidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indomethacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin glucuronide transporter that is inhibited by bilirubin, a fact reported only once in the literature.Conclusions: Data indicate that bilirubin and bilirubin glucuronide are transported into the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter

I. Background: E. coli is a widely used tool for the over-expression of human proteins for studying structure and function. The toxicity of human proteins for E. coli often hampers the expression. This study aims to find conditions for the expression of a membrane transporter known as the carnitine transporter CT2. The knowledge on this transporter is scarce, thus obtaining the recombinant protein is very important for further studies. II. Methods and Results: The cDNAs coding for human CT2 (hCT2) was cloned in the pH6EX3 vector and different transformed E. coli strains were cultured in absence or in presence of glucose. hCT2 expression was obtained. The protein was purified and reconstituted into proteoliposomes in a functionally active state.III. Conclusions: using the appropriate IPTG concentration, together with the addition of glucose, hCT2 has been expressed in E. coli. The protein is active and shows capacity to transport carnitine in proteoliposomes. The results have a great interest in basic biochemistry of membrane transporters and applications to human health since hCT2 is involved in human pathology.

A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae

Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

The Role of Membrane Transporters in Plant Growth and Development, and Abiotic Stress Tolerance

The proteins of membrane transporters (MTs) are embedded within membrane-bounded organelles and are the prime targets for improvements in the efficiency of water and nutrient transportation. Their function is to maintain cellular homeostasis by controlling ionic movements across cellular channels from roots to upper plant parts, xylem loading and remobilization of sugar molecules from photosynthesis tissues in the leaf (source) to roots, stem and seeds (sink) via phloem loading. The plant’s entire source-to-sink relationship is regulated by multiple transporting proteins in a highly sophisticated manner and driven based on different stages of plant growth and development (PG&D) and environmental changes. The MTs play a pivotal role in PG&D in terms of increased plant height, branches/tiller numbers, enhanced numbers, length and filled panicles per plant, seed yield and grain quality. Dynamic climatic changes disturbed ionic balance (salt, drought and heavy metals) and sugar supply (cold and heat stress) in plants. Due to poor selectivity, some of the MTs also uptake toxic elements in roots negatively impact PG&D and are later on also exported to upper parts where they deteriorate grain quality. As an adaptive strategy, in response to salt and heavy metals, plants activate plasma membranes and vacuolar membrane-localized MTs that export toxic elements into vacuole and also translocate in the root’s tips and shoot. However, in case of drought, cold and heat stresses, MTs increased water and sugar supplies to all organs. In this review, we mainly review recent literature from Arabidopsis, halophytes and major field crops such as rice, wheat, maize and oilseed rape in order to argue the global role of MTs in PG&D, and abiotic stress tolerance. We also discussed gene expression level changes and genomic variations within a species as well as within a family in response to developmental and environmental cues.

Membrane Transporters of the Major Facilitator Superfamily Are Essential for Long-Term Maintenance of Phenotypic Tolerance to Multiple Antibiotics in E. coli

We recently showed that the antibiotic-tolerant subpopulation of bacteria or persisters actively maintain the transmembrane proton motive force (PMF) to survive starvation stress for a prolonged period. This work further shows that the reason why antibiotic persisters need to maintain PMF is that PMF is required to support a range of efflux or transportation functions.

Functional Diversity of TonB-Like Proteins in the Heterocyst-Forming Cyanobacterium Anabaena sp. PCC 7120

The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified.