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Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

Summary: Marine diatoms express genes encoding potential phosphate transporter and alkaline phosphatase (APase) under phosphate‐limited (−P) condition. This indicates that diatoms use high‐affinity phosphate uptake system with organic phosphate hydration. The function of molecules playing roles for Pi uptake was determined in this study.Pi uptake and APase activity of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, were monitored during acclimation to −P condition. The transcript levels of Pi transporter were analyzed, and Pi transporters were localized with GFP tagging in diatom cells. KO mutants of plasma membrane solute carrier proteins (SLC34s) or APase were established, and their phenotype was evaluated.Some Na+/Pi transporter candidates, SLC34s in P. tricornutum and T. pseudonana, increased transcript under −P condition. Whole‐cell Pi transport was specifically stimulated by sodium ion but independent of potassium, lithium, or proton. Genome‐editing KO of PtSLC34‐5 and APase (Pt49678) in P. tricornutum was highly inhibitory for Pi uptake, and KO of TpSLC34‐2 was also highly inhibitory for Pi uptake in T. pseudonana.SLC34s and APase were co‐expressed under −P conditions in marine diatoms. SLC34s play a major role in the initial acclimation stage of diatom cells to −P condition and APase plays an increasing role in the prolonged Pi‐starved condition. [ABSTRACT FROM AUTHOR]

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability., Competing Interests: SL, JP, SN, PB No competing interests declared, (© 2024, Lichtinger et al.)

Solute carrier proteins (SLC) are essential membrane transport proteins responsible for transporting lipids, amino acids, sugars, neurotransmitters, and drugs across the biological membranes. Dysfunction of these carrier proteins may lead to an imbalance of biological mechanisms and also in the failure of the transporting pathways of several signaling neurotransmitters. In the present study, a 646 bp of a solute carrier protein (SLC15A4) was cloned and sequenced from the Indian white shrimp, Penaeus indicus. Multiple sequence alignment using ClustalW and phylogenetic analysis of putative SLC15A4 fragment from P. indicus (PiSLC15A4) was performed using Mega X tool. Tissue distribution analysis was carried out using real-time PCR. The differential expressions of PiSLC15A4 were also analyzed in the ovaries and brain tissues of wild-caught female shrimps at different maturation stages and in the brain tissues of captive females subjected to induce maturation by eyestalk ablation. Significant diversity in SLC15A4 sequence obtained from P. indicus was observed when compared to the other species. Tissue distribution analysis confirmed the ubiquitous expression of PiSLC15A4 in all the tissues examined. The differential expressions of PiSLC15A4 indicated higher expression of the gene in brain tissue of females at the vitellogenic stage, while the expressions in ovaries were significantly higher in the immature stage. The differential expressions of PiSLC15A4 in the brain tissues were substantially higher in eyestalk ablated shrimps compared to the eyestalk intact females. The study suggests a role for SLC15A4 in the endocrine signaling pathways stimulating ovarian maturation in P. indicus. [ABSTRACT FROM AUTHOR]

Xenobiotic transport proteins play an important role in determining drug disposition and pharmacokinetics. Our understanding of the role of these important proteins in humans and pre-clinical animal species has increased substantially over the past few decades, and has had an important impact on human medicine; however, veterinary medicine has not benefitted from the same quantity of research into drug transporters in species of veterinary interest. Differences in transporter expression cause difficulties in extrapolation of drug pharmacokinetic parameters between species, and lack of knowledge of species-specific transporter distribution and function can lead to drug–drug interactions and adverse effects. Horses are one species in which little is known about drug transport and transporter protein expression. The purpose of this mini-review is to stimulate interest in equine drug transport proteins and comparative transporter physiology.

As a direct result of intense heat and aridity, deserts are thought to be among the most harsh of environments, particularly for their mammalian inhabitants. Given that osmoregulation can be challenging for these animals, with failure resulting in death, strong selection should be observed on genes related to the maintenance of water and solute balance. One such animal, Peromyscus eremicus, is native to the desert regions of the southwest United States and may live its entire life without oral fluid intake. As a first step toward understanding the genetics that underlie this phenotype, we present a characterization of the P. eremicus transcriptome. We assay four tissues (kidney, liver, brain, testes) from a single individual and supplement this with population level renal transcriptome sequencing from 15 additional animals. We identified a set of transcripts undergoing both purifying and balancing selection based on estimates of Tajima’s D. In addition, we used the branch-site test to identify a transcript—Slc2a9, likely related to desert osmoregulation—undergoing enhanced selection in P. eremicus relative to a set of related non-desert rodents.

Xenobiotic transport proteins play an important role in determining drug disposition and pharmacokinetics. Our understanding of the role of these important proteins in humans and pre-clinical animal species has increased substantially over the past few decades, and has had an important impact on human medicine; however, veterinary medicine has not benefitted from the same quantity of research into drug transporters in species of veterinary interest. Differences in transporter expression cause difficulties in extrapolation of drug pharmacokinetic parameters between species, and lack of knowledge of species-specific transporter distribution and function can lead to drug–drug interactions and adverse effects. Horses are one species in which little is known about drug transport and transporter protein expression. The purpose of this mini-review is to stimulate interest in equine drug transport proteins and comparative transporter physiology. [ABSTRACT FROM AUTHOR]

Lysine methylation is a common posttranslational modification of nuclear and cytoplasmic proteins but is also present in mitochondria. The human protein denoted "family with sequence similarity 173 member B" (FAM173B) was recently uncovered as a mitochondrial lysine (K)-specific methyltransferase (KMT) targeting the c-subunit of mitochondrial ATP synthase (ATPSc), and was therefore renamed ATPSc-KMT. We here set out to investigate the biochemical function of its yet uncharacterized paralogue FAM173A. We demonstrate that FAM173A localizes to mitochondria, mediated by a noncanonical targeting sequence that is partially retained in the mature protein. Immunoblotting analysis using methyllysine-specific antibodies revealed that FAM173A knock-out (KO) abrogates lysine methylation of a single mitochondrial protein in human cells. Mass spectrometry analysis identified this protein as adenine nucleotide translocase (ANT), represented by two highly similar isoforms ANT2 and ANT3. We found that methylation occurs at Lys-52 of ANT, which was previously reported to be trimethylated. Complementation of KO cells with WT or enzyme-dead FAM173A indicated that the enzymatic activity of FAM173A is required for ANT methylation at Lys-52 to occur. Both in human cells and in rat organs, Lys-52 was exclusively trimethylated, indicating that this modification is constitutive, rather than regulatory and dynamic. Moreover, FAM173A-deficient cells displayed increased mitochondrial respiration compared with FAM173A-proficient cells. In summary, we demonstrate that FAM173A is the long-sought KMT responsible for ANT methylation at Lys-52, and point out the functional significance of Lys-52 methylation in ANT. Based on the established naming nomenclature for KMTs, we propose to rename FAM173A to ANT-KMT (gene name ANTKMT )., (© 2019 Małecki et al.)

In beef cattle, proestrus estradiol and subsequent progesterone (P4) concentrations can regulate the endometrial characteristics and thereby determine maternal receptivity toward the embryo. However, the underlying mechanisms linking periovulatory endocrine profiles to receptivity, which is crucial to obtain pregnancy, need to be elucidated. We hypothesized that the size of the preovulatory follicle (POF) and subsequent circulating P4 concentrations, during early diestrus, modulate endometrial levels of glucose transporter transcripts and proteins, and subsequently affect the luminal glucose availability in the uterus. Therefore, follicle growth of Nelore cows was manipulated, and cows were assigned to 2 experimental groups: (1) large follicle and large corpus luteum (LF-LCL) group with a large POF and corpus luteum (CL); and (2) small follicle and small corpus luteum (SF-SCL) group with a small POF and CL. At day 7 post gonadotropin-releasing hormone induced ovulation (gonadotropin-releasing hormone treatment = day 0), animals were slaughtered (n = 18 per group), and uterine tissues and washings were collected for characterization of glucose transporters and glucose levels, respectively. The diameter of POF was larger (P < 0.05) in the LF-LCL cows compared with their SF-SCL counterparts (12.8 ± 0.4 vs 11.1 ± 0.4 mm). Furthermore, CL size (17.49 ± 0.88 vs 14.48 ± 0.52 mm) and circulating P4 concentrations at day 7 (4.5 ± 1.0 vs 3.3 ± 1.1 ng/mL, P < 0.05) were significantly higher in the LF-LCL cows compared with the SF-SCL cows. No differences (P > 0.05) were detected in gene expression patterns of SLC2A1, SLC2A3, SLC2A4, SLC2A5, SLC5A1, ATP1A2, ATP1B2, and SLC37A4. However, the protein abundance of endometrial SLC2A1was increased in the LF-LCL group compared with the SF-SCL group (P < 0.05). SLC2A1 and SLC2A4 protein products were mainly identified at the endometrial luminal and glandular epithelium membranes as well as in the endometrial stroma. Glucose concentrations in uterine washings were similar between groups. In conclusion, we provided information on the potential link between endocrine profiles and glucose transport pathways in the bovine endometrium. More specifically, our data reveal that the size of the POF, and subsequent P4 concentrations, do not functionally affect the main endometrial glucose transporter pathways or uterine fluid glucose concentrations during diestrus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As a direct result of intense heat and aridity, deserts are thought to be among the most harsh of environments, particularly for their mammalian inhabitants. Given that osmoregulation can be challenging for these animals, with failure resulting in death, strong selection should be observed on genes related to the maintenance of water and solute balance. One such animal, Peromyscus eremicus, is native to the desert regions of the southwest United States and may live its entire life without oral fluid intake. As a first step toward understanding the genetics that underlie this phenotype, we present a characterization of the P. eremicus transcriptome. We assay four tissues (kidney, liver, brain, testes) from a single individual and supplement this with population level renal transcriptome sequencing from 15 additional animals. We identified a set of transcripts undergoing both purifying and balancing selection based on estimates of Tajima's D. In addition, we used the branch-site test to identify a transcript-Slc2a9, likely related to desert osmoregulation-undergoing enhanced selection in P. eremicus relative to a set of related non-desert rodents.

Many of the actions of thyroid hormones (THs) occur via TH binding to intracellular receptors. Although it was long thought that THs diffused passively across plasma membranes, it is now recognized that cellular entry is mediated by a variety of membrane transporter proteins. In this study, we identified cDNAs encoding the TH transporters monocarboxylate transferases 8 (mct8) and 10 (mct10) as well as eight distinct organic anion-transporting polypeptide (oatp) proteins from fathead minnow (Pimephales promelas). Analysis of the tissue distribution of transporter mRNAs revealed that mct8 and mct10 transcripts were both abundant in liver, but also present at lower levels in brain, gonad and other tissues. Transcripts encoding oatp1c1 were highly abundant in brain, liver and gonad, and exhibited significant sex differences in the liver and gonad. Treatment of adult male minnows with 3,5,3'-triiodothyronine (T3) or the goitrogen methimazole altered gene transcript abundance for several transporters. Fish given exogenous T3 had reduced mct8 and oapt1c1 mRNA levels in the liver compared to methimazole-treated fish. In the brain, transcripts for mct8, mct10, oatp2b1, and oatp3a1 were each reduced in abundance in fish with elevated T3. As a whole, these results provide evidence that TH status influences the transcriptional dynamics of mct8, mct10 and several Oatp genes including oatp1c1 in teleost fish., (© 2013.)