SLC66 Lysosomal amino acid transporters in GtoPdb v.2021.2

This is a family of 5 evolutionarily related proteins. Their structural similarities suggest that they are transporters. Biochemical evidence supports transporter activity for SLC66A1 (LAAT1) and SLC66A4 (CTNS; Cystinosin). The functions of the 3 remaining members of the family are undetermined

Systematic in silico discovery of novel solute carrier-like proteins from proteomes.

Solute carrier (SLC) proteins represent the largest superfamily of transmembrane transporters. While many of them play key biological roles, their systematic analysis has been hampered by their functional and structural heterogeneity. Based on available nomenclature systems, we hypothesized that many as yet unidentified SLC transporters exist in the human genome, which await further systematic analysis. Here, we present criteria for defining "SLC-likeness" to curate a set of "SLC-like" protein families from the Transporter Classification Database (TCDB) and Protein families (Pfam) databases. Computational sequence similarity searches surprisingly identified ~120 more proteins in human with potential SLC-like properties compared to previous annotations. Interestingly, several of these have documented transport activity in the scientific literature. To complete the overview of the "SLC-ome", we present an algorithm to classify SLC-like proteins into protein families, investigating their known functions and evolutionary relationships to similar proteins from 6 other clinically relevant experimental organisms, and pinpoint structural orphans. We envision that our work will serve as a stepping stone for future studies of the biological function and the identification of the natural substrates of the many under-explored SLC transporters, as well as for the development of new therapeutic applications, including strategies for personalized medicine and drug delivery

SLC66 Lysosomal amino acid transporters in GtoPdb v.2023.1

This is a family of 5 evolutionarily related proteins. Their structural similarities suggest that they are transporters. Biochemical evidence supports transporter activity for SLC66A1 (LAAT1) and SLC66A4 (CTNS; Cystinosin), primarily exporting amino acids from the lysosome to the cytoplasm. The functions of the 3 remaining members of the family are undetermined

Transporter-Mediated Drug Delivery

Transmembrane transport of small organic and inorganic molecules is one of the cornerstones of cellular metabolism. Among transmembrane transporters, solute carrier (SLC) proteins form the largest, albeit very diverse, superfamily with over 400 members. It was recognized early on that xenobiotics can directly interact with SLCs and that this interaction can fundamentally determine their efficacy, including bioavailability and intertissue distribution. Apart from the well-established prodrug strategy, the chemical ligation of transporter substrates to nanoparticles of various chemical compositions has recently been used as a means to enhance their targeting and absorption. In this review, we summarize efforts in drug design exploiting interactions with specific SLC transporters to optimize their therapeutic effects. Furthermore, we describe current and future challenges as well as new directions for the advanced development of therapeutics that target SLC transporters

Redox modulation of STIM-ORAI signaling

AbstractSTIM1 and ORAI1 constitute the core machinery of the ubiquitous store-operated calcium entry pathway and loss of function in these proteins is associated with severe immune and muscular disorders. Other isoforms—STIM1L, STIM2, ORAI2 and ORAI3 exhibit varied expression levels in different cell types along with several other interaction partners and thereby play different roles to facilitate, regulate and fine-tune the calcium entry. STIM proteins convey the Ca2+ store-depletion message to the PM and thereby participate in refilling of the ER by physically interacting with the Ca2+-selective ORAI channels at the PM. STIM and ORAI are exposed to oxidative modifications in the ER, the cytosol, and at the cell surface, and redox-mediated alterations in STIM/ORAI coupling might contribute to autoimmune disorders and cancer progression. This review discusses the redox reactivity of cysteine residues in STIM and ORAI isoforms, focusing on the oxidative modifications of STIM and ORAI proteins by which STIM-ORAI signaling can be modulated

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods

BACKGROUND/AIMS ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different

The Less Well-Known Little Brothers: The SLC9B/NHA Sodium Proton Exchanger Subfamily—Structure, Function, Regulation and Potential Drug-Target Approaches

The SLC9 gene family encodes Na(+)/H(+) exchangers (NHEs), a group of membrane transport proteins critically involved in the regulation of cytoplasmic and organellar pH, cell volume, as well as systemic acid-base and volume homeostasis. NHEs of the SLC9A subfamily (NHE 1–9) are well-known for their roles in human physiology and disease. Much less is known about the two members of the SLC9B subfamily, NHA1 and NHA2, which share higher similarity to prokaryotic NHEs than the SLC9A paralogs. NHA2 (also known as SLC9B2) is ubiquitously expressed and has recently been shown to participate in renal blood pressure and electrolyte regulation, insulin secretion and systemic glucose homeostasis. In addition, NHA2 has been proposed to contribute to the pathogenesis of polycystic kidney disease, the most common inherited kidney disease in humans. NHA1 (also known as SLC9B1) is mainly expressed in testis and is important for sperm motility and thus male fertility, but has not been associated with human disease thus far. In this review, we present a summary of the structure, function and regulation of expression of the SLC9B subfamily members, focusing primarily on the better-studied SLC9B paralog, NHA2. Furthermore, we will review the potential of the SLC9B subfamily as drug targets

Applications of the Microscale Thermophoresis Binding Assay in COVID-19 Research

As the COVID-19 pandemic progresses, new variants of SARS-CoV-2 continue to emerge. This underscores the need to develop optimized tools to study such variants, along with new coronaviruses that may arise in the future. Such tools will also be instrumental in the development of new antiviral drugs. Here, we introduce microscale thermophoresis (MST) as a reliable and versatile tool for coronavirus research, which we demonstrate through three different applications described in this report: (1) binding of the SARS-CoV-2 spike receptor binding domain (RBD) to peptides as a strategy to prevent virus entry, (2) binding of the RBD to the viral receptor ACE2, and (3) binding of the RBD to ACE2 in complex with the amino acid transporter SLC6A20/SIT1 or its allelic variant rs61731475 (p.Ile529Val). Our results demonstrate that MST is a highly precise approach to studying protein–protein and/or protein–ligand interactions in coronavirus research, making it an ideal tool for studying viral variants and developing antiviral agents. Moreover, as shown in our results, a unique advantage of the MST assay over other available binding assays is the ability to measure interactions with membrane proteins in their near-native plasma membrane environment

Functional characterization of a highly specific l-arabinose transporter from Trichoderma reesei

BACKGROUND Lignocellulose biomass has been investigated as a feedstock for second generation biofuels and other value-added products. Some of the processes for biofuel production utilize cellulases and hemicellulases to convert the lignocellulosic biomass into a range of soluble sugars before fermentation with microorganisms such as yeast Saccharomyces cerevisiae. One of these sugars is L-arabinose, which cannot be utilized naturally by yeast. The first step in L-arabinose catabolism is its transport into the cells, and yeast lacks a specific transporter, which could perform this task. RESULTS We identified Trire2_104072 of Trichoderma reesei as a potential L-arabinose transporter based on its expression profile. This transporter was described already in 2007 as D-xylose transporter XLT1. Electrophysiology experiments with Xenopus laevis oocytes and heterologous expression in yeast revealed that Trire2_104072 is a high-affinity L-arabinose symporter with a Km value in the range of [Formula: see text] 0.1-0.2 mM. It can also transport D-xylose but with low affinity (Km [Formula: see text] 9 mM). In yeast, L-arabinose transport was inhibited slightly by D-xylose but not by D-glucose in an assay with fivefold excess of the inhibiting sugar. Comparison with known L-arabinose transporters revealed that the expression of Trire2_104072 enabled yeast to uptake L-arabinose at the highest rate in conditions with low extracellular L-arabinose concentration. Despite the high specificity of Trire2_104072 for L-arabinose, the growth of its T. reesei deletion mutant was only affected at low L-arabinose concentrations. CONCLUSIONS Due to its high affinity for L-arabinose and low inhibition by D-glucose or D-xylose, Trire2_104072 could serve as a good candidate for improving the existing pentose-utilizing yeast strains. The discovery of a highly specific L-arabinose transporter also adds to our knowledge of the primary metabolism of T. reesei. The phenotype of the deletion strain suggests the involvement of other transporters in L-arabinose transport in this species

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a 45Ca2+ uptake assay (7.11 ± 1.93 versus 3.61 ± 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuri