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Principal Investigator: JONATHAN S MARCHANT
Organization: MEDICAL COLLEGE OF WISCONSIN
Fiscal Year: 2024
Award: $448,715
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
Increases in cellular Ca2+ resulting from activation of intracellular Ca2+ channels regulate many physiological
events. Dysfunction of intracellular Ca2+ signaling pathways is involved in many disease states. To appreciate
how cell functions are controlled by Ca2+ signals, and how pathological cues subvert their function, we must
understand how the `functional architecture' of intracellular ion channels controls Ca2+ signaling dynamics.
In this proposal, we investigate the mechanistic basis of Ca2+ release mediated by the second messenger
nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP potently releases Ca2+ from endosomes and
lysosomes by activating a family of cation channels called two-pore channels (TPCs). These signals locally
regulate trafficking of endolysosomal cargoes and can be amplified into whole cell Ca2+ signals by Ca2+-
induced Ca2+ release through neighboring Ca2+ channels on the endoplasmic reticulum (ER). However, the
molecular basis of NAADP-evoked Ca2+ release is unclear as no NAADP binding site has been identified on
the TPC protein. Rather NAADP acts via an unidentified NAADP binding protein (NAADP-BP) associated with
the TPC complex to mediate TPC activation. Here, a multi-PI team (Marchant, Patel, Slama): (i) designed a
novel bifunctional probe to enable a photolabeling strategy that has identified the elusive NAADP-BP as Jupiter
Microtubule Associated Homolog 2 (JPT-2), and (ii) optimized novel TPC activatory ligands that mimic NAADP
action. These tools provide opportunity to finally decipher the molecular basis of NAADP action. This proposal
coalesces medicinal chemistry, molecular analyses, and high-resolution imaging expertise to resolve:
(1) How NAADP binds to JPT2, and how JPT2 associates with TPCs. We will define the nucleotide
binding characteristics of JPT2, and the role of JPT2 in conferring NAADP sensitivity to TPCs
(2) The functional impact of JPT2 on NAADP-evoked Ca2+ signals. We will probe the essentiality and cell
biology of JPT2, resolving its role in endogenous NAADP-evoked Ca2+ signals and targeting of TPCs to
specialized microdomains (membrane contact sites) that couple to endoplasmic reticulum Ca2+ channels.
(3) How NAADP sensitivity is regulated physiologically. Identification of JPT2 provides a molecular
handle to investigate how cellular NAADP sensitivity is physiologically controlled by regulation of JPT2
stability. We show JPT2 and TPCs interact with tankyrase, a poly-ADP-ribosyltransferase. We
hypothesize that JPT2 is a tankyrase substrate, with cellular JPT2 levels controlled by ADP ribosylation.
Terms: <ADP Ribose Transferases><ADP ribosylation><ADP-Ribosyltransferase><ADPRTs><ART Transferases><ARTases><Ablation><Affinity><Antigenic Determinants><Architecture><Autoregulation><Binding><Binding Determinants><Binding Sites><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancers><Cas nuclease technology><Cations><Cell Body><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Signaling><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular biology><Characteristics><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Combining Site><Complex><Coronaviridae><Coronavirus><Cues><Disease><Disorder><Dysfunction><Endoplasmic Reticulum><Endosomes><Engineering / Architecture><Epitopes><Ergastoplasm><Eukaryotic Cell><Event><Exhibits><Family><Family member><Functional disorder><GeneHomolog><Goals><Homeostasis><Homolog><Homologous Gene><Homologue><Human><Intracellular Communication and Signaling><Intracellular Second Messenger><Ion Channel><Ionic Channels><Isoforms><Jupiter><Ligands><Link><Lysosomes><Malignant Neoplasms><Malignant Tumor><Maps><Mediating><Medicinal Chemistry><Membrane><Membrane Channels><Micro-tubule><Microtubules><Modern Man><Molecular><Molecular Analysis><Molecular Interaction><NAADP><NAFLD><Nature><Nucleotides><Obesity><PARP Homolog><Paralysis Agitans><Parkinson><Parkinson Disease><Pathogenicity><Pathologic><Pathology><Pathway interactions><Patients><Permeability><Pharmaceutic Chemistry><Pharmaceutical Chemistry><Phosphate-Binding Proteins><Physiologic><Physiological><Physiological Homeostasis><Physiology><Physiopathology><Play><Primary Parkinsonism><Protein Isoforms><Proteins><Reactive Site><Receptosomes><Recombinants><Regulation><Research><Resolution><Role><Second Messenger Systems><Second Messengers><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Site><Subcellular Process><TRF-1 Interacting, Ankyrin-Related, ADP-Ribose Polymerases><TRF1-Interacting Ankyrin-Related ADP-Ribose Polymerase><Tankyrase><Telomeric Poly-(ADP-Ribose)-Polymerase><Testing><Therapeutic><Viral Diseases><Virus Diseases><Work><adiposity><analog><biological signal transduction><cell biology><cell type><corona virus><corpulence><design><designing><high resolution imaging><imaging approach><imaging based approach><insight><live cell image><live cell imaging><live cellular image><live cellular imaging><malignancy><member><membrane structure><molecular domain><mutant><neoplasm/cancer><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><nicotinate adenine dinucleotide phosphate><nicotinic acid adenine dinucleotide phosphate><non-alcohol fatty liver disease><non-alcoholic fatty liver disease><non-alcoholic liver disease><nonalcoholic fatty liver disease><novel><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><pathophysiology><pathway><pharmacologic><resolutions><response><signal transduction second messengers><small molecule><social role><tool><trafficking><tumor><viral infection><virus infection><virus-induced disease>