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Investigation of Proteins That Associate with Naadp-Gated Two-Pore Channels

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Investigation of Proteins That Associate with Naadp-Gated Two-Pore Channels INVESTIGATION OF PROTEINS THAT ASSOCIATE WITH NAADP-GATED TWO-PORE CHANNELS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Yaping Lin Moshier IN PARTIAL FULTILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor: Dr. Jonathan S. Marchant FEBRUARY, 2012 Copyright 2012 by Yaping Lin Moshier All rights reserved Acknowledgements I would like to acknowledge my advisor Dr. Jonathan Marchant first and foremost, for his full-hearted support and encouragement in the past few years. The work presented here wouldn‟t have been accomplished without his intelligent insights and guidance. I would particularly like to appreciate efforts he put in to help me continue the PhD training, and encourage me to pursue higher achievements. I would like to mention all the current and past members in the Marchant lab for their direct or indirect contributions to this work: Dr. Taisaku Nogi, Dan Zhang, Laramie Rapp, John Chan, Peter Sebastian, Xiaolong Liu, and Mike Keebler. I especially liked scientific discussions and sporadic social events with them. Everyone has made the lab a fun and inspiring working environment. I would also like to thank my committee members- Dr. Stanley Thayer (chair), Dr. Esam El-Fakahany, and Dr. Timothy Walseth- for their great advice, suggestions, and technical assistance to refine this thesis. Direct contributions from other collaborators have also been fascinating, and helped me to achieve my research goals more efficiently. Specifically, I would like to mention the following individuals: Dr. Sandip Patel from the University College London UK for all the TPC constructs and urchin homogenates to perform pharmacological analyses, live cell imaging and interaction studies; Dr. Sean Davidson from UCL for the TPC knockout samples; Dr. James Slama from the University of Toledo for the 5-azido nicotinic acid to make the photoprobe; Dr. Timothy Walseth from the department to synthesize the photoaffinity probe and all the PAL technical support and resources; Dr. Eugen Brailoiu from the Temple University School of Medicine for the calcium release assay in mammalian cells; Todd Markowski, LeeAnn Higgins, and Bruce Witthuhn from the Center for Mass Spectrometry and Proteomics at the U of M for the assistance of MS experimental design and technical support. i During the course of preparing this thesis, my work was mainly supported by the following funding agents: NIH grant GM088790 (Dr. Marchant), BBSRC grant BB/G013721/1 (Dr. Patel), and AHC seed grant from UMN 2101.95 (Dr. Walseth & Dr. Marchant). I was also supported by a Doctoral Dissertation Fellowship from the Graduate School at the University (2010-2011). I would also like to grant special thanks to my family, friends and former co- workers and mentors in the US and Taiwan for their help, support and tremendous belief in me during the hard time in the past five and half years. Last not least, I would like to acknowledge my husband, Adam Moshier, for his understanding that I had to consistently concentrate on study and work, his appreciation and being supportive to my research, tolerating my eccentric scientific talk, and being extremely thoughtful and considerate before and during the graduate training. Without all these, I wouldn‟t have had enough strength to accomplish this work. ii Abstract All living organisms respond to environmental stimuli to coordinate biological activities by eliciting a sequence of signaling cascades, many of which converge to 2+ 2+ regulate cytosolic calcium ion concentration ([Ca ]cyt) via release of intracellular Ca stores. Three agonist-mediated Ca2+ mobilizing 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 2+ mobilizer identified to date, and unlike IP3 and cADPR which target ER Ca stores via 2+ binding to IP3Rs and interaction with RyRs respectively, NAADP-mediated Ca release is evoked from acidic Ca2+ stores, such as endolysosomes and lysosome-like organelles. The endogenous target(s) for NAADP is however controversial, and several Ca2+ channels expressing in the endolysosomal system have been proposed to be gated by NAADP. Recently discovered two-pore channels (TPCs) have been identified as the long-sought NAADP targets via pharmacological, biochemical and genetic approaches. In this thesis, I utilized a photoactivable NAADP analogue, 5-azido NAADP (5N3- NAADP), with the goal of identifying NAADP targets via a direct crosslinking approach coupled with mass spectrometry (MS). My results revealed that 5N3-NAADP was able to recapitulate the basic pharmacological properties of the native ligand NAADP; however, the labeled protein candidate(s) (22/23kDa doublet in mammals and 41kDa in sea urchin) was significantly smaller than the predicted size for TPC proteins. The photolabeled doublet was enriched in soluble fractions in several mammalian cells, even though higher affinity was conserved when membrane bound. The labeling pattern and intensity of the 22/23kDa doublet remained unchanged in either TPC-overexpressing or knockdown samples, suggesting NAADP binding and Ca2+ release are mediated by distinct protein identities. Combinatorial PAL-2D-MS analysis for the high affinity 22/23kDa doublet has identified multiple potential candidates, several of which have been shown to express in both cytosolic and membrane compartments, and are involved in vesicle fusion and trafficking in the endolysosomal system, a physiological mechanism regulated by NAADP and TPCs. Most conservatively, my studies demonstrate that there exists an iii accessory cellular binding partner for NAADP within the TPC signaling complex, which can then serve 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. The second part of my thesis utilized a parallel immunoprecipitation-MS (IP-MS) strategy in an attempt to characterize components of the TPC-signaling complex in mammalian cells. However, while the feasibility of IP-MS strategy to identify TPC proteins was demonstrated, the first candidate examined - CHERP (calcium homeostasis endoplasmic reticulum protein) - failed subsequent validation as a TPC accessory protein. CHERP has been proposed as an integral ER protein that interacts with ryanodine receptors, suggesting an intriguing model for coupling TPCs to RyRs. Remarkably, in contrast to all published data, my results demonstrated that CHERP is actually a nuclear protein. I characterized that the carboxyl terminal region is necessary and sufficient to target CHERP in the nucleus. I identified novel properties for CHERP and proposed its biological functions are to regulate mRNA synthesis, maturation and transport, which may account for some or all of CHERP‟s effect in mediating ER calcium regulation and cellular senescence. This illustrates the need for caution in interpretation of direct (one- step) immunoprecipitation-MS analysis. iv Table of Contents Acknowledgements---------------------------------------------------------------------------i Abstract---------------------------------------------------------------------------------------iii Table of Contents----------------------------------------------------------------------------v Summary of Tables ------------------------------------------------------------------------vi Summary of Figures -----------------------------------------------------------------------vii Summary of Manuscripts-----------------------------------------------------------------viii Abbreviations--------------------------------------------------------------------------------ix Chapter 1: Introduction--------------------------------------------------------------------1 I. Intracellular Ca2+ stores- a growing community of acidic Ca2+ organelles-------------------------------------------------------------------------2 II. NAADP targets acidic Ca2+ stores--------------------------------------------4 III. NAADP targets two-pore channels (TPCs) in the endolysosomal system-----------------------------------------------------------------------------8 IV. Conclusion & Rationale for this study--------------------------------------10 Chapter 2: Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells---------------------------------------------------11 I. Introduction---------------------------------------------------------------------12 II. Materials and Methods--------------------------------------------------------14 III. Results---------------------------------------------------------------------------17 IV. Discussion-----------------------------------------------------------------------35 Chapter 3: Elucidation of the TPC-interactome in mammalian cells by an immunoprecipitation-mass spectrometry strategy-------------------------------------40 I. Introduction---------------------------------------------------------------------41 II. Material and Methods---------------------------------------------------------43 III. Results---------------------------------------------------------------------------47 IV. Discussion-----------------------------------------------------------------------64 Bibliography-------------------------------------------------------------------------------70
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