Browsing by Subject "NAADP"

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    Characterization and modulation of NAADP-dependent calcium signaling events supporting Middle East Respiratory Syndrome Coronavirus infectivity.
    (2018-09) Gunaratne, Gihan
    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-CoV 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 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-CoV fusion with cellular membranes. Pharmacological or genetic inhibition of TPC1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. Following this initial validation of NAADP-gated TPCs supporting pseudotyped MERS-CoV infectivity, a high throughput drug screen of 1534 compounds was conducted in a sea urchin egg homogenate system, yielding 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 strongly correlated with observed inhibition of infectivity of a MERS-CoV pseudovirus. This work highlights the utility of targeting regulators of intracellular trafficking as a novel therapeutic strategy, and provide strong support for the execution of a higher throughput screening campaign using the sea urchin egg homogenate system to discover new ligands for manipulation of NAADP-gated TPC signaling.
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    Investigation of proteins that interact with NAADP-Gated two-pore channels.
    (2012-02) Moshier, Yaping Lin
    All living organisms respond to environmental stimuli by eliciting a sequence of signaling cascades, many of which converge in regulating [Ca2+]cyt via intracellular Ca2+ stores. Three agonist-mediated second messengers have been identified, including inositol 1,4,5-trisphosphate (IP3), cyclic ADP ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP is the most potent calcium mobilizer identified to date, and unlike IP3 and cADPR that target ER Ca2+ stores, NAADP-mediated Ca2+ response is restricted to acidic Ca2+ stores. Several candidate Ca2+ channels expressed in the endolysosomal system have been proposed to be gated by NAADP, with recently two-pore channels (TPCs) emerging as NAADP targets. My research project utilized a radioactive photoactivable NAADP analogue, 32P-5 azido-NAADP (32P-5N3-NAADP), to perform an unbiased assay in a variety of model systems, attempting to verify NAADP targets via a direct crosslinking approach. My results revealed that 5N3-NAADP labeled protein candidate(s) were significantly smaller than predicted sizes of TPC proteins (85-100kDa) in all systems examined (22/23kDa doublet in mammals, 41kDa in sea urchin). Further, the labeling pattern and intensity of the NAADP-targeted doublet remained unchanged in TPC-overexpressing cells. Surprisingly, photoaffinity labeling (PAL) of the high affinity NAADP targets was preserved in TPC-knockout pancreatic samples, further suggesting that NAADP binding and Ca2+ release are mediated by distinct protein identities. My data contradicts current models that TPCs are direct NAADP receptors, implying an alternative cellular binding partner for NAADP on endolysosomes that then serves to indirectly regulate TPC activity. This is an important revision of current dogma, and crucial for rational design of drugs that may modulate NAADP activity. Such therapeutics may be important in disorders (diabetes, lysosomal storage disorders, and neuronal excitotoxicity) where NAADP signaling is pathologically perturbed.