NAADP-binding proteins find their identity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.

Natural variation in the binding pocket of a parasitic flatworm TRPM channel resolves the basis for praziquantel sensitivity.

The drug praziquantel (PZQ) is the key clinical therapy for treating schistosomiasis and other infections caused by parasitic flatworms. A schistosome target for PZQ was recently identified- a transient receptor potential ion channel in the melastatin subfamily (TRPMPZQ)-however, little is known about the properties of TRPMPZQ in other parasitic flatworms. Here, TRPMPZQ orthologs were scrutinized from all currently available parasitic flatworm genomes. TRPMPZQ is present in all parasitic flatworms, and the consensus PZQ binding site was well conserved. Functional profiling of trematode, cestode, and a free-living flatworm TRPMPZQ ortholog revealed differing sensitives (~300-fold) of these TRPMPZQ channels toward PZQ, which matched the varied sensitivities of these different flatworms to PZQ. Three loci of variation were defined across the parasitic flatworm TRPMPZQ pocketome with the identity of an acidic residue in the TRP domain acting as a gatekeeper residue impacting PZQ residency within the TRPMPZQ ligand binding pocket. In trematodes and cyclophyllidean cestodes, which display high sensitivity to PZQ, this TRP domain residue is an aspartic acid which is permissive for potent activation by PZQ. However, the presence of a glutamic acid residue found in other parasitic and free-living flatworm TRPMPZQ was associated with lower sensitivity to PZQ. The definition of these different binding pocket architectures explains why PZQ shows high therapeutic effectiveness against specific fluke and tapeworm infections and will help the development of better tailored therapies toward other parasitic infections of humans, livestock, and fish.

The anthelmintic meclonazepam activates a schistosome transient receptor potential channel.

Parasitic flatworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood flukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain-binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

Progesterone receptor membrane component 1 facilitates Ca2+ signal amplification between endosomes and the endoplasmic reticulum.

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.

Molecular and cellular basis of praziquantel action in the cardiovascular system.

The anthelmintic drug praziquantel (PZQ) causes contraction of parasitic schistosomes as well as constriction of blood vessels within the mesenteric vasculature of the host where the adult blood flukes reside. The contractile action of PZQ on the vasculature is mediated by the activation of host serotonergic 5-HT2B receptors (5-HT2BRs). However, the molecular basis for PZQ interaction with these targets and the location of these 5-HT2B receptors in the vessel wall have not been experimentally defined. Evaluation of a PZQ docking pose within the 5-HT2BR orthosteric site, using both Ca2+ reporter and bioluminescence resonance energy transfer (BRET) assays, identified residues F3406.51 and F3416.52 (transmembrane helix 6, TM6) as well as L209EL2 (extracellular loop 2) as critical for PZQ-mediated agonist activity. A key determinant of PZQ selectivity for the 5-HT2B receptor over the 5-HT2A/2C receptors was determined by M2185.39 in transmembrane helix 5 (TM5) of the orthosteric site. Mutation of this residue to valine (M218V), as found in 5-HT2A and 5-HT2C, decreased PZQ agonist activity, whereas the reciprocal mutation (V215M) in 5-HT2C increased PZQ activity. Two-photon imaging in intact mesenteric arterial strips visualized PZQ-evoked Ca2+ transients within the smooth muscle cells of the vessel wall. PZQ also triggered cytoplasmic Ca2+ signals in arterial smooth muscle cells in primary culture that were isolated from mesenteric blood vessels. These data define the molecular basis for PZQ action on 5-HT2B receptors localized in vascular smooth muscle.

Endo-Lysosomal Two-Pore Channels and Their Protein Partners.

Two-pore channels are ion channels expressed on acidic organelles such as the various vesicles that constitute the endo-lysosomal system. They are permeable to Ca2+ and Na+ and activated by the second messenger NAADP as well as the phosphoinositide, PI(3,5)P2 and/or voltage. Here, we review the proteins that interact with these channels including recently identified NAADP receptors.

Synthesis and biological evaluation of novel photo-clickable adenosine and cyclic ADP-ribose analogs: 8-N3-2'-O-propargyladenosine and 8-N3-2'-O-propargyl-cADPR.

A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2'-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.

Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs.

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca2+ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2'-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N3-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.

Enteropathogenic Escherichia coli Infection Inhibits Intestinal Ascorbic Acid Uptake via Dysregulation of Its Transporter Expression.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) infection causes prolonged, watery diarrhea leading to morbidity and mortality. Although EPEC infection impacts nutrient transporter function and expression in intestinal epithelial cells, the effects of EPEC infection on intestinal absorption of ascorbic acid (AA) have not yet been investigated. AIMS: To investigate the effect of EPEC infection on intestinal AA uptake process and expression of both AA transporters. METHODS: We used two experimental models: human-derived intestinal epithelial Caco-2 cells and mice. 14C-AA uptake assay, Western blot, RT-qPCR, and promoter assay were performed. RESULTS: EPEC (WT) as well as ΔespF and ΔespG/G2 mutant-infected Caco-2 cells showed markedly inhibited AA uptake, while other mutants (ΔescN, ΔespA, ΔespB, and ΔespD) did not affect AA uptake. Infection also reduced protein and mRNA expression levels for both hSVCT1 and hSVCT2. EPEC-infected mice showed marked inhibitory effect on AA uptake and decreased protein and mRNA expression levels for both mSVCT1 and mSVCT2 in jejunum and colon. MicroRNA regulators of SVCT1 and SVCT2 (miR103a, miR141, and miR200a) were upregulated significantly upon EPEC infection in both Caco-2 and mouse jejunum and colon. In addition, expression of the accessory protein glyoxalate reductase/hydroxypyruvate reductase (GRHPR), which regulates SVCT1 function, was markedly decreased by EPEC infection in both models. CONCLUSIONS: These findings suggest that EPEC infection causes inhibition in AA uptake through a multifactorial dysregulation of SVCT1 and SVCT2 expression in intestinal epithelial cells.

Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake.

Vitamin C (ascorbic acid: AA) uptake in neurons occurs via the sodium-dependent vitamin C transporter-2 (SVCT2), which is highly expressed in the central nervous system (CNS). During chronic neuroinflammation or infection, CNS levels of lipopolysaccharide (LPS) and LPS-induced tumor necrosis factor-α (TNFα) are increased. Elevated levels of LPS and TNFα have been associated with neurodegenerative diseases together with reduced levels of AA. However, little is known about the impacts of LPS and TNFα on neuronal AA uptake. The objective of this study was to examine the effect of LPS and TNFα on SVCT2 expression and function using in vitro and in vivo approaches. Treatment of SH-SY5Y cells with either LPS or TNFα inhibited AA uptake. This reduced uptake was associated with a significant decrease in SVCT2 protein and mRNA levels. In vivo exposure to LPS or TNFα also decreased SVCT2 protein and mRNA levels in mouse brains. Both LPS and TNFα decreased SLC23A2 promoter activity. Further, the inhibitory effect of LPS on a minimal SLC23A2 promoter was attenuated when either the binding site for the transcription factor Sp1 was mutated or cells were treated with the NF-κB inhibitor, celastrol. We conclude that inflammatory signals suppress AA uptake by impairing SLC23A2 transcription through opposing regulation of Sp1 and NF-κB factors.

The anthelmintic drug praziquantel activates a schistosome transient receptor potential channel.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca2+ influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use. Here, we demonstrate that PZQ activates a schistosome transient receptor potential (TRP) channel, christened SmTRPMPZQ, present in parasitic schistosomes and other PZQ-sensitive parasites. Several properties of SmTRPMPZQ were consistent with known effects of PZQ on schistosomes, including (i) nanomolar sensitivity to PZQ; (ii) stereoselectivity toward (R)-PZQ; (iii) mediation of sustained Ca2+ signals in response to PZQ; and (iv) a pharmacological profile that mirrors the well-known effects of PZQ on muscle contraction and tegumental disruption. We anticipate that these findings will spur development of novel therapeutic interventions to manage schistosome infections and broader interest in PZQ, which is finally unmasked as a potent flatworm TRP channel activator.

Enterotoxigenic Escherichia coli heat labile enterotoxin inhibits intestinal ascorbic acid uptake via a cAMP-dependent NF-κB-mediated pathway.

Vitamin C is an antioxidant and acts as a cofactor for many enzymatic reactions. Humans obtain vitamin C from dietary sources via intestinal absorption, a process that involves the sodium-dependent vitamin C transporters-1 and -2 (SVCT1 and SVCT2). Enterotoxigenic Escherichia coli (ETEC) infection impacts intestinal absorption/secretory functions, but nothing is known about its effect on ascorbic acid (AA) uptake. Here we demonstrate that infection of Caco-2 cells with ETEC led to a significant inhibition in intestinal AA uptake. This inhibition was associated with a marked reduction in hSVCT1 and hSVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression levels as well as significant inhibition in the activity of both the SLC23A1 and SLC23A2 promoters. Similarly, exposure of mice to ETEC led to a significant inhibition in intestinal AA uptake and reduction in mSVCT1 and mSVCT2 protein, mRNA, and hnRNA expression levels. Inhibition was caused by the action of heat labile enterotoxin (LT), since infecting Caco-2 cells with LT-deficient ETEC (ΔLT) failed to impact AA uptake. Because LT activates adenylate cyclase, we also examined the effect of dibutyryl-cAMP in AA uptake by Caco-2 cells and observed a significant inhibition. Furthermore, treating the cells with celastrol, a specific NF-κB inhibitor, significantly blocked the inhibition of AA uptake caused by ETEC infection. Together, these data demonstrate that ETEC infection impairs intestinal AA uptake through a cAMP-dependent NF-κB-mediated pathway that regulates both SLC23A1 and SLC23A2 transcription. NEW & NOTEWORTHY Our findings demonstrate that heat-labile enterotoxin produced by enterotoxigenic Escherichia coli inhibits AA uptake in intestinal epithelial cells and mouse intestine. This effect is mediated through transcriptional repression of SLC23A1 (SVCT1) and SLC23A2 (SVCT2) via a cAMP-dependent NF-κB signaling pathway.

Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca2+ channel in neuromuscular function and regeneration.

The robust regenerative capacity of planarian flatworms depends on the orchestration of signaling events from early wounding responses through the stem cell enacted differentiative outcomes that restore appropriate tissue types. Acute signaling events in excitable cells play an important role in determining regenerative polarity, rationalized by the discovery that sub-epidermal muscle cells express critical patterning genes known to control regenerative outcomes. These data imply a dual conductive (neuromuscular signaling) and instructive (anterior-posterior patterning) role for Ca2+ signaling in planarian regeneration. Here, to facilitate study of acute signaling events in the excitable cell niche, we provide a de novo transcriptome assembly from the planarian Dugesia japonica allowing characterization of the diverse ionotropic portfolio of this model organism. We demonstrate the utility of this resource by proceeding to characterize the individual role of each of the planarian voltage-operated Ca2+ channels during regeneration, and demonstrate that knockdown of a specific voltage operated Ca2+ channel (Cav1B) that impairs muscle function uniquely creates an environment permissive for anteriorization. Provision of the full transcriptomic dataset should facilitate further investigations of molecules within the planarian voltage-gated channel portfolio to explore the role of excitable cell physiology on regenerative outcomes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Inhibition of intestinal ascorbic acid uptake by lipopolysaccharide is mediated via transcriptional mechanisms.

Ascorbic acid (AA) accumulation in intestinal epithelial cells is an active transport process mainly mediated by two sodium-dependent vitamin C transporters (SVCT-1 and SVCT-2). To date, little is known about the effect of gut microbiota generated lipopolysaccharide (LPS) on intestinal absorption of water-soluble vitamins. Therefore, the objective of this study was to investigate the effects of bacterially-derived LPS on AA homeostasis in enterocytes using Caco-2 cells, mouse intestine and intestinal enteroids models. Pre-treating Caco-2 cells and mice with LPS led to a significant decrease in carrier-mediated AA uptake. This inhibition was associated with a significant reduction in SVCT-1 and SVCT-2 protein, mRNA, and hnRNA expression. Furthermore, pre-treating enteroids with LPS also led to a marked decrease in SVCT-1 and SVCT-2 protein and mRNA expression. Inhibition of SVCT-1 and SVCT-2 occurred at least in part at the transcriptional level as promoter activity of SLC23A1 and SLC23A2 was attenuated following LPS treatment. Subsequently, we examined the protein and mRNA expression levels of HNF1α and Sp1 transcription factors, which are needed for basal SLC23A1 and SLC23A2 promoter activity, and found that they were significantly decreased in the LPS treated Caco-2 cells and mouse jejunum; this was reflected on level of the observed reduction in the interaction of these transcription factors with their respective promoters in Caco-2 cells treated with LPS. Our findings indicate that LPS inhibits intestinal carrier- mediated AA uptake by down regulating the expression of both vitamin C transporters and transcriptional regulation of SLC23A1 and SLC23A2 genes.

Tumor necrosis factor alpha reduces intestinal vitamin C uptake: a role for NF-κB-mediated signaling.

Sodium-dependent vitamin C transporter-1 (SVCT-1) is the major transporter mediating intestinal vitamin C uptake. Intestinal inflammation and prolonged infection are associated with increased serum and intestinal mucosa levels of tumor necrosis factor-α (TNF-α), which also exerts profound effects on the intestinal absorption process. Elevated levels of TNF-α have been linked to the pathogenesis of inflammatory bowel disease (IBD) and malabsorption of nutrients, and patients with this condition have low levels of vitamin C. To date, little is known about the effect of TNF-α on intestinal absorption of vitamin C. We studied the impact of TNF-α on ascorbic acid (AA) transport using a variety of intestinal preparations. The expression level of human SVCT-1 mRNA is significantly lower in patients with IBD. TNF-α treated Caco-2 cells and mice showed a significant inhibition of intestinal 14C-AA uptake. This inhibition was associated with significant decreases in SVCT-1 protein, mRNA, and heterogeneous nuclear RNA levels in TNF-α treated Caco-2 cells, mouse jejunum, and enteroids. Also, TNF-α caused a significant inhibition in the SLC23A1 promoter activity. Furthermore, treatment of Caco-2 cells with celastrol (NF-κB inhibitor) blocked the inhibitory effect caused by TNF-α on AA uptake, SVCT-1 protein, and mRNA expression, as well as the activity of SLC23A1 promoter. Treatment of TNF-α also led to a significant decrease in the expression of hepatocyte nuclear factor-1-α, which drives the basal activity of SLC23A1 promoter, and this effect was reversed by celastrol. Together, these findings show that TNF-α inhibits intestinal AA uptake, and this effect is mediated, at least in part, at the level of transcription of the SLC23A1 gene via the NF-κB pathway. NEW & NOTEWORTHY Our findings show that tumor necrosis factor-α inhibits intestinal ascorbic acid uptake in both in vitro and in vivo systems, and this inhibitory effect is mediated, at least in part, at the level of transcription of the SLC23A1 (sodium-dependent vitamin C transporter-1) gene via the NF-κB pathway.

A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors.

Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets ('target selection') and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics.

Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract.

Mammalian cells utilize two transporters for the uptake of ascorbic acid (AA), Na+-dependent vitamin C transporter SVCT-1 and SVCT-2. In the intestine, these transporters are involved in AA absorption and are expressed at the apical and basolateral membrane domains of the polarized epithelia, respectively. Little is known about the differential expression of these two transporters along the anterior-posterior axis of the intestinal tract and the molecular mechanism(s) that dictate this pattern of expression. We used mouse and human intestinal cDNAs to address these issues. The results showed a significantly lower rate of carrier-mediated AA uptake by mouse colon than jejunum. This was associated with a significantly lower level of expression of SVCT-1 and SVCT-2 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels in the colon than the jejunum, implying the involvement of transcriptional mechanism(s). Similarly, expression levels of SVCT-1 and SVCT-2 mRNA and hnRNA were significantly lower in human colon. We also examined the levels of expression of hepatocyte nuclear factor 1α and specificity protein 1, which drive transcription of the Slc23a1 and Slc23a2 promoters, respectively, and found them to be markedly lower in the colon. Furthermore, significantly lower levels of the activating markers for histone (H3) modifications [H3 trimethylation of lysine 4 (H3K4me3) and H3 triacetylation of lysine 9 (H3K9ac)] were observed in the Slc23a1 and Slc23a2 promoters in the colon. These findings show, for the first time, that SVCT-1 and SVCT-2 are differentially expressed along the intestinal tract and that this pattern of expression is, at least in part, mediated via transcriptional/epigenetic mechanisms.NEW & NOTEWORTHY Our findings show, for the first time, that transporters of the water-soluble vitamin ascorbic acid (i.e., the vitamin C transporters SVCT-1 and SVCT-2) are differentially expressed along the length of the intestinal tract and that the pattern of expression is mediated, at least in part, by transcriptional and epigenetic mechanism(s) affecting both Slc23a1 and Slc23a2 genes.

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.

Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease.

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation.

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca(2+) homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.

'Death and axes': unexpected Ca²âº entry phenologs predict new anti-schistosomal agents.

Schistosomiasis is a parasitic flatworm disease that infects 200 million people worldwide. The drug praziquantel (PZQ) is the mainstay therapy but the target of this drug remains ambiguous. While PZQ paralyses and kills parasitic schistosomes, in free-living planarians PZQ caused an unusual axis duplication during regeneration to yield two-headed animals. Here, we show that PZQ activation of a neuronal Ca²âº channel modulates opposing dopaminergic and serotonergic pathways to regulate 'head' structure formation. Surprisingly, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. These divergent outcomes (death versus axis duplication) result from the same Ca²âº entry mechanism, and comprise unexpected Ca²âº phenologs with meaningful predictive value. Surprisingly, basic research into axis patterning mechanisms provides an unexpected route for discovering novel antischistosomal agents.