Publications by authors named "Timothy Walseth"

56 Publications

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

Sci Signal 2021 Mar 23;14(675). Epub 2021 Mar 23.

The Ca Signalling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

NAADP-evoked Ca release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca 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 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca microdomains and delayed the onset and decreased the amplitude of global Ca 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 Ca release from the endoplasmic reticulum through RYR.
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http://dx.doi.org/10.1126/scisignal.abd5647DOI Listing
March 2021

Essential requirement for JPT2 in NAADP-evoked Ca signaling.

Sci Signal 2021 03 23;14(675). Epub 2021 Mar 23.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca 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 Ca 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 Ca signaling and control of coronaviral entry.
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http://dx.doi.org/10.1126/scisignal.abd5605DOI Listing
March 2021

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

Bioorg Med Chem 2021 01 1;30:115901. Epub 2020 Dec 1.

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, United States. Electronic address:

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca 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-N-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.
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http://dx.doi.org/10.1016/j.bmc.2020.115901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869823PMC
January 2021

Novel Pathway of Adenosine Generation in the Lungs from NAD: Relevance to Allergic Airway Disease.

Molecules 2020 Oct 27;25(21). Epub 2020 Oct 27.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA.

Adenosine and uric acid (UA) play a pivotal role in lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). In the present experiments, we measured adenosine synthesis from nicotinamide adenine dinucleotide (NAD) in membranes prepared from wild type (WT) and CD38 knockout (CD38KO) mouse lungs, from cultured airway smooth muscle and epithelial cells, and in bronchoalveolar lavage fluid after airway challenge with epidemiologically relevant allergens. Adenosine was determined using an enzymatically coupled assay that produces ATP and is detected by luminescence. Uric acid was determined by ELISA. Exposure of cultured airway epithelial cells to extract caused significant nucleotide (NAD and ATP) release in the culture media. The addition of NAD to membranes prepared from WT mice resulted in faster generation of adenosine compared to membranes from CD38KO mice. Formation of adenosine from NAD affected UA and ATP concentrations, its main downstream molecules. Furthermore, NAD and adenosine concentrations in the bronchoalveolar lavage fluid decreased significantly following airway challenge with house-dust mite extract in WT but not in CD38KO mice. Thus, NAD is a significant source of adenosine and UA in the airways in mouse models of allergic airway disease, and the capacity for their generation from NAD is augmented by CD38, a major NADase with high affinity for NAD. This novel non-canonical NAD-adenosine-UA pathway that is triggered by allergens has not been previously described in the airways.
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http://dx.doi.org/10.3390/molecules25214966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663290PMC
October 2020

Role of CD38/cADPR signaling in obstructive pulmonary diseases.

Curr Opin Pharmacol 2020 04 29;51:29-33. Epub 2020 May 29.

Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, United States.

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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http://dx.doi.org/10.1016/j.coph.2020.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529733PMC
April 2020

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

Cell Calcium 2019 11 1;83:102060. Epub 2019 Aug 1.

Department of Medicinal and Biological Chemistry, University of Toledo College of Pharmacy and Pharmaceutical Sciences, 3000 Arlington Avenue, Toledo, OH, 43614, United States. Electronic address:

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca mobilizing second messenger which triggers Ca 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 Ca 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 Ca 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 Ca release. In mammalian cell homogenates, incubation with low concentrations of [P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [P]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 [P]5-N-NAADP.
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http://dx.doi.org/10.1016/j.ceca.2019.102060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774857PMC
November 2019

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

Sci Signal 2019 01 29;12(566). Epub 2019 Jan 29.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA.

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.
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http://dx.doi.org/10.1126/scisignal.aau5378DOI Listing
January 2019

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

Biochim Biophys Acta Mol Cell Res 2019 07 4;1866(7):1180-1188. Epub 2018 Dec 4.

Department of Pharmacology, University of Minnesota Medical School, 312 Church St., Minneapolis, MN 55455-0217, United States of America. Electronic address:

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca 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 5N-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca responses. Higher concentrations of 5N-8-ethynyl were required to elicit Ca release or displace P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of P-5N-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 P-5N-NAADP, the clickable P-5N-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 P-5N-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N-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.
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http://dx.doi.org/10.1016/j.bbamcr.2018.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101546PMC
July 2019

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

Cell Calcium 2018 11 16;75:42-52. Epub 2018 Aug 16.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee WI 53226, USA. Electronic address:

The Ca 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 Ca 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 Ca 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 Ca release (but not cADPR- or IP-evoked Ca 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 Ca release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca release without depleting acidic Ca 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 Ca 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 IP-dependent Ca signaling with potential therapeutic value.
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http://dx.doi.org/10.1016/j.ceca.2018.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286156PMC
November 2018

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

Cell Calcium 2018 11 9;75:30-41. Epub 2018 Aug 9.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee WI 53226, USA. Electronic address:

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, Ca-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 Ca 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 Ca 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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http://dx.doi.org/10.1016/j.ceca.2018.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251489PMC
November 2018

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

Mediators Inflamm 2018 7;2018:8942042. Epub 2018 Feb 7.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA.

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 (Ca) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca 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.
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http://dx.doi.org/10.1155/2018/8942042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821947PMC
September 2018

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

Pharmacol Ther 2017 Apr 7;172:116-126. Epub 2016 Dec 7.

Department of Veterinary and Biomedical Sciences, University of Minnesota at Twin Cities, USA. Electronic address:

CD38 is an ectoenzyme that catalyzes the conversion of β-nicotinamide adenine dinucleotide (β-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.
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http://dx.doi.org/10.1016/j.pharmthera.2016.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346344PMC
April 2017

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

J Biol Chem 2016 Feb 4;291(9):4503-22. Epub 2016 Jan 4.

From the Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, and

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.
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http://dx.doi.org/10.1074/jbc.M115.681833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813477PMC
February 2016

TPC1 Knockout Knocks Out TPC1.

Mol Cell Biol 2015 May;35(10):1882-3

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, USA

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http://dx.doi.org/10.1128/MCB.00020-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405651PMC
May 2015

Expression of Ca²⁺-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

EMBO J 2015 Jul 14;34(13):1743-58. Epub 2015 Apr 14.

Department of Pharmacology, University of Oxford, Oxford, UK

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.
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http://dx.doi.org/10.15252/embj.201490009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516428PMC
July 2015

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca²⁺ Release.

J Med Chem 2015 Apr 13;58(8):3593-610. Epub 2015 Apr 13.

†Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, 3000 Arlington Avenue, Toledo Ohio 43614, United States.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480638PMC
April 2015

CD38 and airway hyper-responsiveness: studies on human airway smooth muscle cells and mouse models.

Can J Physiol Pharmacol 2015 Feb 9;93(2):145-53. Epub 2014 Dec 9.

a Department of Surgical & Radiological Sciences, University of California, Davis, CA 95616, USA.

Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.
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http://dx.doi.org/10.1139/cjpp-2014-0410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648235PMC
February 2015

MicroRNA-708 regulates CD38 expression through signaling pathways JNK MAP kinase and PTEN/AKT in human airway smooth muscle cells.

Respir Res 2014 Aug 31;15:107. Epub 2014 Aug 31.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, 1971 Commonwealth Avenue, St, Paul 55108, MN, USA.

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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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http://dx.doi.org/10.1186/s12931-014-0107-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156970PMC
August 2014

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

Proc Natl Acad Sci U S A 2014 Sep 25;111(36):13087-92. Epub 2014 Aug 25.

Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455;

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.
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http://dx.doi.org/10.1073/pnas.1407004111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246952PMC
September 2014

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: difference in specificity between human and sea urchin NAADP receptors.

Cell Calcium 2014 Feb 28;55(2):93-103. Epub 2013 Dec 28.

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo Health Sciences Campus, 3000 Arlington Avenue, Toledo, OH 43614, United States. Electronic address:

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure-activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel "caged" 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca2+ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.
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http://dx.doi.org/10.1016/j.ceca.2013.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015339PMC
February 2014

Altered CD38/Cyclic ADP-Ribose Signaling Contributes to the Asthmatic Phenotype.

J Allergy (Cairo) 2012 20;2012:289468. Epub 2012 Nov 20.

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.

CD38 is a transmembrane glycoprotein expressed in airway smooth muscle cells. The enzymatic activity of CD38 generates cyclic ADP-ribose from β-NAD. Cyclic ADP-ribose mobilizes intracellular calcium during activation of airway smooth muscle cells by G-protein-coupled receptors through activation of ryanodine receptor channels in the sarcoplasmic reticulum. Inflammatory cytokines that are implicated in asthma upregulate CD38 expression and increase the calcium responses to contractile agonists in airway smooth muscle cells. The augmented intracellular calcium responses following cytokine exposure of airway smooth muscle cells are inhibited by an antagonist of cyclic ADP-ribose. Airway smooth muscle cells from CD38 knockout mice exhibit attenuated intracellular calcium responses to agonists, and these mice have reduced airway response to inhaled methacholine. CD38 also contributes to airway hyperresponsiveness as shown in mouse models of allergen or cytokine-induced inflammatory airway disease. In airway smooth muscle cells obtained from asthmatics, the cytokine-induced CD38 expression is significantly enhanced compared to expression in cells from nonasthmatics. This differential induction of CD38 expression in asthmatic airway smooth muscle cells stems from increased activation of MAP kinases and transcription through NF-κB, and altered post-transcriptional regulation through microRNAs. We propose that increased capacity for CD38 signaling in airway smooth muscle in asthma contributes to airway hyperresponsiveness.
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http://dx.doi.org/10.1155/2012/289468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3508580PMC
December 2012

miR-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells.

Am J Physiol Lung Cell Mol Physiol 2012 Sep 6;303(5):L460-8. Epub 2012 Jul 6.

Dept. of Veterinary and Biomedical Sciences, College of Veterinary Medicine, Univ. of Minnesota, St. Paul, MN 55108, USA.

CD38, a membrane protein expressed in airway smooth muscle (ASM) cells, plays a role in cellular Ca(2+) dynamics and ASM contractility. In human ASM (HASM) cells, TNF-α induces CD38 expression through activation of MAPKs, NF-κB, and AP-1, and its expression is differentially elevated in cells from asthmatic patients compared with cells from nonasthmatic subjects. The CD38 3'-untranslated region (UTR) has targets for miR-140-3p. We hypothesized that miR-140-3p regulates CD38 expression in HASM cells by altering CD38 mRNA stability. Basal and TNF-α-induced expression of miR-140-3p was determined in nonasthmatic ASM (NAASM) and asthmatic ASM (AASM) cells. NAASM and AASM cells were transfected with control, miR-140-3p mimic, or miR-140-3p antagomirs, and CD38 expression and CD38 mRNA stability were determined. Luciferase reporter assays were used to determine miR-140-3p binding to the CD38 3'-UTR. Activation of p38, ERK, and JNK MAPKs, NF-κB, and AP-1 was determined in miR-140-3p mimic-transfected NAASM. TNF-α attenuated miR-140-3p expression in NAASM and AASM cells, but at a greater magnitude in AASM cells. CD38 mRNA expression was attenuated by miR-140-3p mimic at comparable magnitude in NAASM and AASM cells. Mutated miR-140-3p target on the CD38 3'-UTR reversed the inhibition of luciferase activity by miR-140-3p mimic. CD38 mRNA stability was unaltered by miR-140-3p mimic in NAASM or AASM cells following arrest of transcription. TNF-α-induced activation of p38 MAPK and NF-κB was attenuated by miR-140-3p mimic. The findings indicate that miR-140-3p modulates CD38 expression in HASM cells through direct binding to the CD38 3'-UTR and indirect mechanisms involving activation of p38 MAPK and NF-κB. Furthermore, indirect mechanisms appear to play a major role in the regulation of CD38 expression.
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http://dx.doi.org/10.1152/ajplung.00041.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468424PMC
September 2012

The Molecular Basis for Ca Signalling by NAADP: Two-Pore Channels in a Complex?

Messenger (Los Angel) 2012 Jun;1(1):63-76

Department of Cell and Developmental Biology, University College London, WC1E 6BT, UK.

NAADP is a potent Ca mobilizing messenger in a variety of cells but its molecular mechanism of action is incompletely understood. Accumulating evidence indicates that the poorly characterized two-pore channels (TPCs) in animals are NAADP sensitive Ca-permeable channels. TPCs localize to the endo-lysosomal system but are functionally coupled to the better characterized endoplasmic reticulum Ca channels to generate physiologically relevant complex Ca signals. Whether TPCs directly bind NAADP is not clear. Here we discuss the idea based on recent studies that TPCs are the pore-forming subunits of a protein complex that includes tightly associated, low molecular weight NAADP-binding proteins.
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http://dx.doi.org/10.1166/msr.2012.1003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4193953PMC
June 2012

Nicotinic Acid Adenine Dinucleotide 2'-Phosphate (NAADP) Binding Proteins in T-Lymphocytes.

Messenger (Los Angel) 2012 Jun;1(1):86-94

The Calcium Signalling Group, Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany ; Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Although several channels, including two-pore channels (TPC), ryanodine receptor (RYR) and mucolipin (TRP-ML1) have been implicated in NAADP regulation of calcium signaling, the NAADP receptor has not been identified. In this study, the photoaffinity probe, [P]-5-azido-NAADP ([P]-5-N-NAADP), was used to study NAADP binding proteins in extracts from NAADP responsive Jurkat T-lymphocytes. [P]-5-N-NAADP photolabeling of Jurkat S100 cytosolic fractions resulted in the labeling of at least ten distinct proteins. Several of these S100 proteins, including a doublet at 22/23 kDa and small protein at 15 kDa displayed selectivity for NAADP as the labeling was protected by inclusion of unlabeled NAADP, whereas the structurally similar NADP required much higher concentrations for protection. Interestingly, the labeling of several S100 proteins (60, 45, 33 and 28 kDa) was stimulated by low concentrations of unlabeled NAADP, but not by NADP. The effect of NAADP on the labeling of the 60 kDa protein was biphasic, peaking at 100 nM with a five-fold increase and displaying no change at 1 µM NAADP. Several proteins were also photolabeled when the P100 membrane fraction from Jurkat cells was examined. Similar to the results with S100, a 22/23 kDa doublet and a 15 kDa protein appeared to be selectively labeled. NAADP did not increase the labeling of any P100 proteins as it did in the S100 fraction. The photolabeled S100 and P100 proteins were successfully resolved by two-dimensional gel electrophoresis. [P]-5-N-NAADP photolabeling and two-dimensional electrophoresis should represent a suitable strategy in which to identify and characterize NAADP binding proteins.
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http://dx.doi.org/10.1166/msr.2012.1008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017354PMC
June 2012

Regulation of CD38 expression in human airway smooth muscle cells: role of class I phosphatidylinositol 3 kinases.

Am J Respir Cell Mol Biol 2012 Oct 3;47(4):427-35. Epub 2012 May 3.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA.

The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.
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http://dx.doi.org/10.1165/rcmb.2012-0025OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488627PMC
October 2012

Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

J Biol Chem 2012 Jan 23;287(4):2308-15. Epub 2011 Nov 23.

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

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 [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP 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.
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http://dx.doi.org/10.1074/jbc.M111.306563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268392PMC
January 2012

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells.

J Biol Chem 2012 Jan 23;287(4):2296-307. Epub 2011 Nov 23.

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) 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 ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) 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.
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http://dx.doi.org/10.1074/jbc.M111.305813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268391PMC
January 2012

HIV-1 and IL-1β regulate astrocytic CD38 through mitogen-activated protein kinases and nuclear factor-κB signaling mechanisms.

J Neuroinflammation 2011 Oct 25;8:145. Epub 2011 Oct 25.

Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

Background: Infection with human immunodeficiency virus type-1 (HIV)-1 leads to some form of HIV-1-associated neurocognitive disorders (HAND) in approximately half of the cases. The mechanisms by which astrocytes contribute to HIV-1-associated dementia (HAD), the most severe form of HAND, still remain unresolved. HIV-1-encephalitis (HIVE), a pathological correlate of HAD, affects an estimated 9-11% of the HIV-1-infected population. Our laboratory has previously demonstrated that HIVE brain tissues show significant upregulation of CD38, an enzyme involved in calcium signaling, in astrocytes. We also reported an increase in CD38 expression in interleukin (IL)-1β-activated astrocytes. In the present investigation, we studied regulatory mechanisms of CD38 gene expression in astrocytes activated with HIV-1-relevant stimuli. We also investigated the role of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in astrocyte CD38 regulation.

Methods: Cultured human astrocytes were transfected with HIV-1(YU-2) proviral clone and levels of CD38 mRNA and protein were measured by real-time PCR gene expression assay, western blot analysis and immunostaining. Astrocyte activation by viral transfection was determined by analyzing proinflammatory chemokine levels using ELISA. To evaluate the roles of MAPKs and NF-κB in CD38 regulation, astrocytes were treated with MAPK inhibitors (SB203580, SP600125, U0126), NF-κB interfering peptide (SN50) or transfected with dominant negative IκBα mutant (IκBαM) prior to IL-1β activation. CD38 gene expression and CD38 ADP-ribosyl cyclase activity assays were performed to analyze alterations in CD38 levels and function, respectively.

Results: HIV-1(YU-2)-transfection significantly increased CD38 mRNA and protein expression in astrocytes (p < 0.01) in a dose-dependent manner and induced astrocyte activation. IL-β-activation of HIV-1(YU-2)-transfected astrocytes significantly increased HIV-1 gene expression (p < 0.001). Treatment with MAPK inhibitors or NF-κB inhibitor SN50 abrogated IL-1β-induced CD38 expression and activity in astrocytes without altering basal CD38 levels (p < 0.001). IκBαM transfection also significantly inhibited IL-1β-mediated increases in CD38 expression and activity in astrocytes (p < 0.001).

Conclusion: The present findings demonstrate a direct involvement of HIV-1 and virus-induced proinflammatory stimuli in regulating astrocyte-CD38 levels. HIV-1(YU-2)-transfection effectively induced HIV-1p24 protein expression and activated astrocytes to upregulate CCL2, CXCL8 and CD38. In astrocytes, IL-1β-induced increases in CD38 levels were regulated through the MAPK signaling pathway and by the transcription factor NF-κB. Future studies may be directed towards understanding the role of CD38 in response to infection and thus its role in HAND.
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http://dx.doi.org/10.1186/1742-2094-8-145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247131PMC
October 2011

Gain of function of cardiac ryanodine receptor in a rat model of type 1 diabetes.

Cardiovasc Res 2011 Jul 18;91(2):300-9. Epub 2011 Mar 18.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800, USA.

Aims: Ventricular myocytes isolated from hearts of streptozotocin (STZ)-diabetic rats exhibit increased spontaneous Ca(2+) release. Studies attribute this defect to an enhancement in activity of type 2 ryanodine receptor (RyR2). To date, underlying reasons for RyR2 dysregulation remain undefined. This study assesses whether the responsiveness of RyR2 following stimulation by intrinsic ligands is being altered during experimental type 1 diabetes (T1D).

Methods And Results: M-mode echocardiography established a cardiomyopathy in 8 weeks STZ-diabetic rats. Confocal microscopy confirmed an increase in the spontaneous Ca(2+) release in isolated ventricular myocytes. Western blots revealed no significant change in steady-state levels of the RyR2 protein. When purified to homogeneity and incorporated into planar lipid bilayers, RyR2 from STZ-diabetic rats (dRyR2) exhibited reduced current amplitude at ±35 mV. dRyR2 was also more responsive to intrinsic cytoplasmic activators Ca(2+), adenosine triphosphate, and cyclic adenosine diphosphate ribose and less responsive to the cytoplasmic deactivator Mg(2+). Threshold for the activation of RyR2 by trans (luminal) Ca(2+) was also reduced. These changes were independent of phosphorylation at Ser2808 and Ser2814. Two weeks of insulin treatment starting after 6 weeks of diabetes blunted the phenotype change, indicating that the gain of function is specific to the diabetes and not the result of STZ interacting directly with RyR2.

Conclusion: These data show, for the first time, that RyR2 is acquiring a gain-of-function phenotype independent of its phosphorylation status during T1D and provides new insights for the enhanced spontaneous Ca(2+) release in myocytes from T1D rats.
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http://dx.doi.org/10.1093/cvr/cvr076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125072PMC
July 2011

TNF-α regulation of CD38 expression in human airway smooth muscle: role of MAP kinases and NF-κB.

Adv Exp Med Biol 2011 ;691:449-59

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA.

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http://dx.doi.org/10.1007/978-1-4419-6612-4_46DOI Listing
April 2011