Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca
from intracellular stores in a wide variety of ...cell types, tissues and organisms. Current evidence suggests that NAADP may function as a trigger to initiate a Ca
signal that is then amplified by other Ca
release mechanisms. A fundamental question that remains unanswered is the identity of the NAADP receptor. Our recent studies have identified HN1L/JPT2 as a high affinity NAADP binding protein that is essential for the modulation of Ca
channels.
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•Nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from acidic organelles.•Middle East Respiratory Syndrome coronavirus (MERS-CoV) traffics through host-cell acidic ...organelles.•Blockers of NAADP action inhibited pseudotyped MERS-CoV infectivity.•Knockdown of two-pore channels (TPCs), a target of NAADP, also blocked MERS-CoV infectivity.
Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. Identifying novel targets/pathways required for MERS infectivity is therefore important for developing novel therapeutics. As an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of MERS-CoV. In this context, Ca2+-permeable channels within the endolysosomal system regulate both the luminal environment and trafficking events, meriting investigation of their role in regulating processing and trafficking of MERS-CoV. Knockdown of endogenous two-pore channels (TPCs), targets for the Ca2+ mobilizing second messenger NAADP, impaired infectivity in a MERS-CoV spike pseudovirus particle translocation assay. This effect was selective as knockdown of the lysosomal cation channel mucolipin-1 (TRPML1) was without effect. Pharmacological inhibition of NAADP-evoked Ca2+ release using several bisbenzylisoquinoline alkaloids also blocked MERS pseudovirus translocation. Knockdown of TPC1 (biased endosomally) or TPC2 (biased lysosomally) decreased the activity of furin, a protease which facilitates MERS fusion with cellular membranes. Pharmacological or genetic inhibition of TPC1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. Overall, these data support a selective, spatially autonomous role for TPCs within acidic organelles to support MERS-CoV translocation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca2+ from intracellular acidic Ca2+ stores. Recent evidence has identified the two-pore ...channels (TPCs) within the endolysosomal system as NAADP-regulated Ca2+ channels that release organellar Ca2+ in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP (32P-5N3NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca2+ signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N3-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca2+ release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.
Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) activates two-pore channels (TPCs) to release Ca2+ from intracellular acidic Ca2+ stores.
Results: A photoactivatable probe based on NAADP labels proteins distinct from TPCs.
Conclusion: NAADP may bind to an accessory protein within a larger TPC complex.
Significance: First evidence that TPCs act as NAADP-activated Ca2+ release channels, but not NAADP receptors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Significance Two-pore channels (TPCs) are a recently discovered family of endolysosomal ion channels, but their regulation is controversial. By defining the TPC interactome, we provide a community ...resource that illuminates TPC complex regulation and resolves associations with novel partners and processes. Physical interactions with endolysosomal trafficking regulators predominate, and Rab GTPases impart isoform-specific roles for TPCs in organelle proliferation and cellular pigmentation. These data imply a fundamental role for TPCs in trafficking that augurs significance for disease states exhibiting lysosomal proliferation where TPC dysregulation may drive pathogenesis.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as ...endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized 32P-5-azidonicotinic acid adenine dinucleotide phosphate (32P-5N3NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with 32P-5N3NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. 32P-5N3NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. 32P-5N3NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by 32P-5N3NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.
Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates calcium release from internal acidic stores via two-pore channels (TPCs).
Results: A novel photosensitive probe (5-azido-NAADP) identified high affinity NAADP binding sites that interact with, but are distinct from, TPCs.
Conclusion: High affinity NAADP-binding proteins complex with TPCs.
Significance: This work provides new mechanistic insights into how NAADP regulates calcium release via TPCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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The worldwide socioeconomical burden associated with chronic respiratory diseases is substantial. Enzymes involved in the metabolism of nicotinamide adenine dinucleotide (NAD) are ...increasingly being implicated in chronic airway diseases. One such enzyme, CD38, utilizes NAD to produce several metabolites, including cyclic ADP ribose (cADPR), which is involved in calcium signaling in airway smooth muscle (ASM). Upregulation of CD38 in ASM caused by exposure to cytokines or allergens leads to enhanced calcium mobilization by agonists and the development of airway hyperresponsiveness (AHR) to contractile agonists. Glucocorticoids and microRNAs can suppress CD38 expression in ASM, whereas cADPR antagonists such as 8Br-cADPR can directly antagonize intracellular calcium mobilization. Bronchodilators act via CD38-independent mechanisms. CD38-dependent mechanisms could be developed for chronic airway diseases therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The cell-surface protein CD38 mediates airway smooth muscle (ASM) contractility by generating cyclic ADP-ribose, a calcium-mobilizing molecule. In human ASM cells, TNF-α augments CD38 expression ...transcriptionally by NF-κB and AP-1 activation and involving MAPK and PI3K signaling. CD38-/- mice develop attenuated airway hyperresponsiveness following allergen or cytokine challenge. The post-transcriptional regulation of CD38 expression in ASM is relatively less understood. In ASM, microRNAs (miRNAs) regulate inflammation, contractility, and hyperproliferation. The 3' Untranslated Region (3'UTR) of CD38 has multiple miRNA binding sites, including a site for miR-708. MiR-708 is known to regulate PI3K/AKT signaling and hyperproliferation of other cell types. We investigated miR-708 expression, its regulation of CD38 expression and the underlying mechanisms involved in such regulation in human ASM cells.
Growth-arrested human ASM cells from asthmatic and non-asthmatic donors were used. MiRNA and mRNA expression were measured by quantitative real-time PCR. CD38 enzymatic activity was measured by a reverse cyclase assay. Total and phosphorylated MAPKs and PI3K/AKT as well as enzymes that regulate their activation were determined by Western blot analysis of cell lysates following miRNA transfection and TNF-α stimulation. Dual luciferase reporter assays were performed to determine whether miR-708 binds directly to CD38 3'UTR to alter gene expression.
Using target prediction algorithms, we identified several miRNAs with potential CD38 3'UTR target sites and determined miR-708 as a potential candidate for regulation of CD38 expression based on its expression and regulation by TNF-α. TNF-α caused a decrease in miR-708 expression in cells from non-asthmatics while it increased its expression in cells from asthmatics. Dual luciferase reporter assays in NIH-3 T3 cells revealed regulation of expression by direct binding of miR-708 to CD38 3'UTR. In ASM cells, miR-708 decreased CD38 expression by decreasing phosphorylation of JNK MAPK and AKT. These effects were associated with increased expression of MKP-1, a MAP kinase phosphatase and PTEN, a phosphatase that terminates PI3 kinase signaling.
In human ASM cells, TNF-α-induced CD38 expression is regulated by miR-708 directly binding to 3'UTR and indirectly by regulating JNK MAPK and PI3K/AKT signaling and has the potential to control airway inflammation, ASM contractility and proliferation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The second messenger NAADP triggers Ca2+ release from endo‐lysosomes. Although two‐pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating ...the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAADP‐dependent Ca2+ responses as assessed by single‐cell Ca2+ imaging or patch‐clamp of single endo‐lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2−/− cells, NAADP sensitivity was restored by re‐expressing wild‐type TPCs, but not by mutant versions with impaired Ca2+‐permeability, nor by TRPML1. Another mouse line formerly reported as TPC‐null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP‐induced Ca2+ release. High‐affinity 32PNAADP binding still occurs in Tpcn1/2−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+‐permeable channels indispensable for NAADP signalling.
Synopsis
By presenting a new double‐knockout mouse model this study demonstrates that two‐pore channels (TPCs) are responsible for NAADP‐dependent Ca2+ release from endo‐lysosomes.
Endogenous two‐pore channels (TPCs) are essential for NAADP‐stimulated Ca2+ currents and Ca2+ release.
Re‐expression of TPCs in validated null‐background cells restores NAADP sensitivity.
Truncated TPC1 and TPC2 proteins—potentially expressed in other reported TPC knockouts—are still functional.
TPCs are not required for high‐affinity NAADP‐binding.
PI(3,5)P2 is a promiscuous modulator of endo‐lysosomal channels.
By presenting a new double‐knockout mouse model this study demonstrates that two‐pore channels (TPCs) are responsible for NAADP‐dependent Ca2+ release from endo‐lysosomes.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•The photoactive and clickable adenosine analog, 8-N3-2ʹ-O-propargyladenosine was synthesized from 8-bromoadenosine in three steps.•Photo-clickable adenosine is a useful new scaffold ...for chemical probes to identify proteins which interact with a variety of adenosine nucleosides and nucleotides.•8-N3-2ʹ-O-propargyladenosine was converted to its NAD derivative, and enzymatically cyclized using Aplasia californica ADP-ribosyl cyclase. The result was a photoactive and clickable analog of the second messenger cyclic ADP-ribose.•Combining 8‑adenosyl and 2ʹ-O-ribosyl substituents into a cyclic ADP-ribose analog was shown to result in a significant decrease in agonist potency when compared to the potency of the two singly substituted analogs.
A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2ʹ-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2ʹ-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5ʹ-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2ʹ-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2ʹ-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. 2ʹ-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-N3-2ʹ-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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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