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.

Convergent activation of two-pore channels mediated by the NAADP-binding proteins JPT2 and LSM12.

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) evokes calcium ion (Ca2+) release from endosomes and lysosomes by activating two-pore channels (TPCs) on these organelles. Rather than directly binding to TPCs, NAADP associates with proteins that indirectly confer NAADP sensitivity to the TPC complex. We investigated whether and how the NAADP-binding proteins Jupiter microtubule-associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12) contributed to NAADP-TPC-Ca2+ signaling in human cells. Biochemical and functional analyses revealed that recombinant JPT2 and LSM12 both bound to NAADP with high affinity and that endogenous JPT2 and LSM12 independently associated with TPC1 and TPC2. On the basis of knockout and rescue analyses, both NAADP-binding proteins were required to support NAADP-evoked Ca2+ signaling and contributed to endolysosomal trafficking of pseudotyped coronavirus particles. These data reveal that the NAADP-binding proteins JPT2 and LSM12 convergently regulate NAADP-evoked Ca2+ release and function through TPCs.

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.

Identification of a dihydropyridine scaffold that blocks ryanodine receptors.

Ryanodine receptors (RyRs) are large, intracellular ion channels that control Ca2+ release from the sarco/endoplasmic reticulum. Dysregulation of RyRs in skeletal muscle, heart, and brain has been implicated in various muscle pathologies, arrhythmia, heart failure, and Alzheimer's disease. Therefore, there is considerable interest in therapeutically targeting RyRs to normalize Ca2+ homeostasis in scenarios involving RyR dysfunction. Here, a simple invertebrate screening platform was used to discover new chemotypes targeting RyRs. The approach measured Ca2+ signals evoked by cyclic adenosine 5'-diphosphate ribose, a second messenger that sensitizes RyRs. From a 1,534-compound screen, FLI-06 (currently described as a Notch "inhibitor") was identified as a potent blocker of RyR activity. Two closely related tyrosine kinase inhibitors that stimulate and inhibit Ca2+ release through RyRs were also resolved. Therefore, this simple screen yielded RyR scaffolds tractable for development and revealed an unexpected linkage between RyRs and trafficking events in the early secretory pathway.

NAADP: From Discovery to Mechanism.

Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca2+ 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 Ca2+ signal that is then amplified by other Ca2+ 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 Ca2+ channels.

Essential requirement for JPT2 in NAADP-evoked Ca2+ signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.

HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.

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.

Role of CD38/cADPR signaling in obstructive pulmonary diseases.

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.

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This 'all-in-one-clickable' NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP.

The pseudokinase domains of guanylyl cyclase-A and -B allosterically increase the affinity of their catalytic domains for substrate.

Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity. Molecular modeling indicated that all the residues of the ATP-binding site of the prototypical kinase PKA, except the catalytic aspartate, are conserved in the PKDs of GC-A and GC-B. Kinase-inactivating alanine substitutions for the invariant lysine in subdomain II or the aspartate in the DYG-loop of GC-A and GC-B failed to decrease enzyme phosphate content, consistent with the PKDs lacking kinase activity. In contrast, both mutations reduced enzyme activation by blocking the ability of ATP to decrease the Michaelis constant without affecting peptide-dependent activation. The analogous lysine-to-alanine substitution in a glutamate-substituted phosphomimetic mutant form of GC-B also reduced enzyme activity, consistent with ATP stimulating guanylyl cyclase activity through an allosteric, phosphorylation-independent mechanism. Mutations designed to rigidify the conserved regulatory or catalytic spines within the PKDs increased guanylyl cyclase activity, increased sensitivity to natriuretic peptide, or reduced the Michaelis constant in the absence of ATP, consistent with ATP binding stabilizing the PKD in a conformation analogous to that of catalytically active kinases. We conclude that allosteric mechanisms evolutionarily conserved in the PKDs promote the catalytic activation of transmembrane guanylyl cyclases.

5-Azido-8-ethynyl-NAADP: A bifunctional, clickable photoaffinity probe for the identification of NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca2+ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a 'clickable' ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N3-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca2+ responses. Higher concentrations of 5N3-8-ethynyl were required to elicit Ca2+ release or displace 32P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of 32P-5N3-8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional 32P-5N3-NAADP, the clickable 32P-5N3-8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the 'clickable' ethynyl moiety. No proteins were specifically labeled by 32P-5N3-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N3-8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a 'bifunctional' probe with utility for isolating NAADP-binding proteins.

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells.

The Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca2+ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca2+ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca2+ release (but not cADPR- or IP3-evoked Ca2+ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen 'hits' exhibiting >80% inhibition of NAADP-evoked Ca2+ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca2+ release without depleting acidic Ca2+ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca2+ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP3-dependent Ca2+ signaling with potential therapeutic value.

NAADP-dependent Ca2+ signaling regulates Middle East respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system.

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.

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms.

Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed.

CD38 in the pathogenesis of allergic airway disease: Potential therapeutic targets.

CD38 is an ectoenzyme that catalyzes the conversion of ß-nicotinamide adenine dinucleotide (ß-NAD) to cyclic adenosine diphosphoribose (cADPR) and adenosine diphosphoribose (ADPR) and NADP to nicotinic acid adenine dinucleotide phosphate (NAADP) and adenosine diphosphoribose-2'-phosphate (ADPR-P). The metabolites of NAD and NADP have roles in calcium signaling in different cell types including airway smooth muscle (ASM) cells. In ASM cells, inflammatory cytokines augment CD38 expression and to a greater magnitude in cells from asthmatics, indicating a greater capacity for the generation of cADPR and ADPR in ASM from asthmatics. CD38 deficient mice develop attenuated airway responsiveness to inhaled methacholine following allergen sensitization and challenge compared to wild-type mice indicating its potential role in asthma. Regulation of CD38 expression in ASM cells is achieved by mitogen activated protein kinases, specific isoforms of PI3 kinases, the transcription factors NF-κB and AP-1, and post-transcriptionally by microRNAs. This review will focus on the role of CD38 in intracellular calcium regulation in ASM, contribution to airway inflammation and airway hyperresponsiveness in mouse models of allergic airway inflammation, the transcriptional and post-transcriptional mechanisms of regulation of expression, and outline approaches to inhibit its expression and activity.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca²âº Release.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20-500-fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca(2+) with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced.

Expression of Ca²âº-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

The second messenger NAADP triggers Ca(2+) 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 Ca(2+) responses as assessed by single-cell Ca(2+) 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 Ca(2+)-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 Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.