Role of solute carrier transporters in regulating dendritic cell maturation and function.

Dendritic cells (DCs) are specialized antigen-presenting cells that initiate and regulate innate and adaptive immune responses. Solute carrier (SLC) transporters mediate diverse physiological functions and maintain cellular metabolite homeostasis. Recent studies have highlighted the significance of SLCs in immune processes. Notably, upon activation, immune cells undergo rapid and robust metabolic reprogramming, largely dependent on SLCs to modulate diverse immunological responses. In this review, we explore the central roles of SLC proteins and their transported substrates in shaping DC functions. We provide a comprehensive overview of recent studies on amino acid transporters, metal ion transporters, and glucose transporters, emphasizing their essential contributions to DC homeostasis under varying pathological conditions. Finally, we propose potential strategies for targeting SLCs in DCs to bolster immunotherapy for a spectrum of human diseases.

Membrane transporters in cell physiology, cancer metabolism and drug response.

By controlling the passage of small molecules across lipid bilayers, membrane transporters influence not only the uptake and efflux of nutrients, but also the metabolic state of the cell. With more than 450 members, the Solute Carriers (SLCs) are the largest transporter super-family, clustering into families with different substrate specificities and regulatory properties. Cells of different types are, therefore, able to tailor their transporter expression signatures depending on their metabolic requirements, and the physiological importance of these proteins is illustrated by their mis-regulation in a number of disease states. In cancer, transporter expression is heterogeneous, and the SLC family has been shown to facilitate the accumulation of biomass, influence redox homeostasis, and also mediate metabolic crosstalk with other cell types within the tumour microenvironment. This Review explores the roles of membrane transporters in physiological and malignant settings, and how these roles can affect drug response, through either indirect modulation of sensitivity or the direct transport of small-molecule therapeutic compounds into cells.

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Targeting SLC transporters: small molecules as modulators and therapeutic opportunities.

Solute carrier (SLCs) transporters mediate the transport of a broad range of solutes across biological membranes. Dysregulation of SLCs has been associated with various pathologies, including metabolic and neurological disorders, as well as cancer and rare diseases. SLCs are therefore emerging as key targets for therapeutic intervention with several recently approved drugs targeting these proteins. Unlocking this large and complex group of proteins is essential to identifying unknown SLC targets and developing next-generation SLC therapeutics. Recent progress in experimental and computational techniques has significantly advanced SLC research, including drug discovery. Here, we review emerging topics in therapeutic discovery of SLCs, focusing on state-of-the-art approaches in structural, chemical, and computational biology, and discuss current challenges in transporter drug discovery.

The Structure and Mechanism of Drug Transporters.

Drug transporters are integral membrane proteins that play a critical role in drug disposition by affecting absorption, distribution, and excretion. They translocate drugs, as well as endogenous molecules and toxins, across membranes using ATP hydrolysis, or ion/concentration gradients. In general, drug transporters are expressed ubiquitously, but they function in drug disposition by being concentrated in tissues such as the intestine, the kidneys, the liver, and the brain. Based on their primary sequence and their mechanism, transporters can be divided into the ATP-binding cassette (ABC), solute-linked carrier (SLC), and the solute carrier organic anion (SLCO) superfamilies. Many X-ray crystallography and cryo-electron microscopy (cryo-EM) structures have been solved in the ABC and SLC transporter superfamilies or of their bacterial homologs. The structures have provided valuable insight into the structural basis of transport. This chapter will provide particular focus on the promiscuous drug transporters because of their effect on drug disposition and the challenges associated with them.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

Disruption of small molecule transporter systems by Transporter-Interfering Chemicals (TICs).

Small molecule transporters (SMTs) in the ABC and SLC families are important players in disposition of diverse endo- and xenobiotics. Interactions of environmental chemicals with these transporters were first postulated in the 1990s, and since validated in numerous in vitro and in vivo scenarios. Recent results on the co-crystal structure of ABCB1 with the flame-retardant BDE-100 demonstrate that a diverse range of man-made and natural toxic molecules, hereafter termed transporter-interfering chemicals (TICs), can directly bind to SMTs and interfere with their function. TIC-binding modes mimic those of substrates, inhibitors, modulators, inducers, and possibly stimulants through direct and allosteric mechanisms. Similarly, the effects could directly or indirectly agonize, antagonize or perhaps even prime the SMT system to alter transport function. Importantly, TICs are distinguished from drugs and pharmaceuticals that interact with transporters in that exposure is unintended and inherently variant. Here, we review the molecular mechanisms of environmental chemical interaction with SMTs, the methodological considerations for their evaluation, and the future directions for TIC discovery.

A unique ferrous iron binding mode is associated with large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Releasing of iron from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore-binding protein respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind to a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa.

The Druggability of Solute Carriers.

The transport of materials across membranes is a vital process for all aspects of cellular function, including growth, metabolism, and communication. Protein transporters are the molecular gates that control this movement and serve as key points of regulation for these processes, thus representing an attractive class of therapeutic targets. With more than 400 members, the solute carrier (SLC) membrane transport proteins are the largest family of transporters, yet, they are pharmacologically underexploited relative to other protein families and many of the available chemical tools possess suboptimal selectivity and efficacy. Fortuitously, there is increased interest in elucidating the physiological roles of SLCs as well as growing recognition of their therapeutic potential. This Perspective provides an overview of the SLC superfamily, including their biochemical and functional features, as well as their roles in various human diseases. In particular, we explore efforts and associated challenges toward drugging SLCs, as well as highlight opportunities for future drug discovery.

Functional analysis of glycosylation using Drosophila melanogaster.

The glycosylation of proteins and lipids has various essential roles in a diverse range of biological processes, including embryogenesis, organ development, neurogenesis, maintenance of homeostasis, immune response, and tumorigenesis. Drosophila melanogaster is one of the representative multicellular model organisms, which have many useful genetic manipulation tools; it is used in developmental biology as well as classical and molecular genetics. Glycobiology is not an exception and many studies using Drosophila have been performed in this field to clarify novel functions of glycans. Recently, genome-wide screening and functional analyses were performed in whole body, wings, eyes, neuromuscular junctions, and immune organs. Furthermore, detailed studies with Drosophila mutants of glycosyltransferases, nucleotide sugar transporters, and glycosidases revealed novel functions of N-linked glycans, glycosaminoglycans, glycolipids, and O-linked glycans including mucin type O-glycan, O-Fuc, O-Man, and O-GlcNAc. As many of these functions are common between Drosophila and humans, these mutants represent good models for human disease. In this review, recent studies of glycan functions using Drosophila are summarized.

Advances and Challenges in Rational Drug Design for SLCs.

There are over 420 human solute carrier (SLC) transporters from 65 families that are expressed ubiquitously in the body. The SLCs mediate the movement of ions, drugs, and metabolites across membranes and their dysfunction has been associated with a variety of diseases, such as diabetes, cancer, and central nervous system (CNS) disorders. Thus, SLCs are emerging as important targets for therapeutic intervention. Recent technological advances in experimental and computational biology allow better characterization of SLC pharmacology. Here we describe recent approaches to modulate SLC transporter function, with an emphasis on the use of computational approaches and computer-aided drug design (CADD) to study nutrient transporters. Finally, we discuss future perspectives in the rational design of SLC drugs.

Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters.

1. Organophosphorus pesticides (OPs) are known to interact with human ATP-binding cassette drug efflux pumps. The present study was designed to determine whether they can also target activities of human solute carrier (SLC) drug transporters. 2. The interactions of 13 OPs with SLC transporters involved in drug disposition, such as organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs), were mainly investigated using transporter-overexpressing cell clones and fluorescent or radiolabeled reference substrates. 3. With a cut-off value of at least 50% modulation of transporter activity by 100 µM OPs, OAT1 and MATE2-K were not impacted, whereas OATP1B1 and MATE1 were inhibited by two and three OPs, respectively. OAT3 activity was similarly blocked by three OPs, and was additionally stimulated by one OP. Five OPs cis-stimulated OATP2B1 activity. Both OCT1 and OCT2 were inhibited by the same eight OPs, including fenamiphos and phosmet, with IC50 values however in the 3-30 µM range, likely not relevant to environmental exposure. 4. These data demonstrated that various OPs inhibit SLC drug transporter activities, especially those of OCT1 and OCT2, but only when used at high concentrations not expected to occur in environmentally-exposed humans.

An Aspartate-Specific Solute-Binding Protein Regulates Protein Kinase G Activity To Control Glutamate Metabolism in Mycobacteria.

Signaling by serine/threonine phosphorylation controls diverse processes in bacteria, and identification of the stimuli that activate protein kinases is an outstanding question in the field. Recently, we showed that nutrients stimulate phosphorylation of the protein kinase G substrate GarA in Mycobacterium smegmatis and Mycobacterium tuberculosis and that the action of GarA in regulating central metabolism depends upon whether it is phosphorylated. Here we present an investigation into the mechanism by which nutrients activate PknG. Two unknown genes were identified as co-conserved and co-expressed with PknG: their products were a putative lipoprotein, GlnH, and putative transmembrane protein, GlnX. Using a genetic approach, we showed that the membrane protein GlnX is functionally linked to PknG. Furthermore, we determined that the ligand specificity of GlnH matches the amino acids that stimulate GarA phosphorylation. We determined the structure of GlnH in complex with different amino acid ligands (aspartate, glutamate, and asparagine), revealing the structural basis of ligand specificity. We propose that the amino acid concentration in the periplasm is sensed by GlnH and that protein-protein interaction allows transmission of this information across the membrane via GlnX to activate PknG. This sensory system would allow regulation of nutrient utilization in response to changes in nutrient availability. The sensor, signaling, and effector proteins are conserved throughout the Actinobacteria, including the important human pathogen Mycobacterium tuberculosis, industrial amino acid producer Corynebacterium glutamicum, and antibiotic-producing Streptomyces species.IMPORTANCE Tuberculosis (TB) kills 5,000 people every day, and the prevalence of multidrug-resistant TB is increasing in every country. The processes by which the pathogen Mycobacterium tuberculosis senses and responds to changes in its environment are attractive targets for drug development. Bacterial metabolism differs dramatically between growing and dormant cells, and these changes are known to be important in pathogenesis of TB. Here, we used genetic and biochemical approaches to identify proteins that allow M. tuberculosis to detect amino acids in its surroundings so that it can regulate its metabolism. We have also shown how individual amino acids are recognized. The findings have broader significance for other actinobacterial pathogens, such as nontuberculous mycobacteria, as well as Actinobacteria used to produce billions of dollars of amino acids and antibiotics every year.

SLC6 Transporter Folding Diseases and Pharmacochaperoning.

The human genome encodes 19 genes of the solute carrier 6 (SLC6) family; non-synonymous changes in the coding sequence give rise to mutated transporters, which are misfolded and thus cause diseases in the affected individuals. Prominent examples include mutations in the transporters for dopamine (DAT, SLC6A3), for creatine (CT1, SLC6A8), and for glycine (GlyT2, SLC6A5), which result in infantile dystonia, mental retardation, and hyperekplexia, respectively. Thus, there is an obvious unmet medical need to identify compounds, which can remedy the folding deficit. The pharmacological correction of folding defects was originally explored in mutants of the serotonin transporter (SERT, SLC6A4), which were created to study the COPII-dependent export from the endoplasmic reticulum. This led to the serendipitous discovery of the pharmacochaperoning action of ibogaine. Ibogaine and its metabolite noribogaine also rescue several disease-relevant mutants of DAT. Because the pharmacology of DAT and SERT is exceptionally rich, it is not surprising that additional compounds have been identified, which rescue folding-deficient mutants. These compounds are not only of interest for restoring DAT function in the affected children. They are also likely to serve as useful tools to interrogate the folding trajectory of the transporter. This is likely to initiate a virtuous cycle: if the principles underlying folding of SLC6 transporters are understood, the design of pharmacochaperones ought to be facilitated.

Potentiating SLC transporter activity: Emerging drug discovery opportunities.

Maintaining the integrity of cellular membranes is critical to protecting metabolic activities and genetic information from the environment. Regulation of transport across membranes of essential chemicals, including water, nutrients, hormones and many drugs, is therefore key to cellular homeostasis and physiological processes. The two main transporter superfamilies are ATP-binding cassette (ABC) transporters that primarily function as efflux transporters, and the solute carrier (SLC) transporters. SLC transporters encompass 52 gene families with almost 400 different human transporter genes. Although long under-explored, SLC transporters are an emerging drug target class and the molecular target of several approved inhibitor drugs, such as selective serotonin reuptake inhibitors (SSRIs) for depression and sodium/glucose co-transporter (SGLT2) inhibitors for diabetes. Interestingly though, although loss-of-function mutations in numerous human SLC transporters are linked to Mendelian diseases, few reports of SLC transporter activators have appeared, and only inhibitors have been advanced to clinical studies. In this commentary, we discuss several strategies for potentiating SLC transporter function, from direct acting potentiators to modulators of transcription, translation or trafficking. We review the progress made in recent years toward the understanding of the structural and molecular basis of SLC transporter function and the pathways and mechanisms that regulate SLC expression, and describe the opportunities these new insights present for discovery of SLC transporter potentiators. Finally, we highlight the challenges associated with the various approaches and provide some thoughts on future directions that might facilitate the search for SLC potentiators with therapeutic potential.

Classification Systems of Secondary Active Transporters.

Membrane-bound solute carrier (SLC) transporter proteins are vital to the human body, as they sustain homeostasis by moving soluble molecule as nutrients, drugs, and waste across lipid membranes. Of the 430 identified secondary active transporters in humans, 30% are still orphans, and systematic research has been requested to elaborate on their possible involvement in diseases and their potential as drug targets. To enable this, the various classification systems in use must be understood and used correctly. In this review, we describe how various classification systems for human SLCs are constructed, and how they overlap and differ. To facilitate communication between researchers and to avoid ambiguities, everyone must clearly state which classification system they are referring to when writing scientific articles.