Mitochondrial Uptake of Thiamin Pyrophosphate: Physiological and Cell Biological Aspects (vol 8, e73503, 2013)

[This corrects the article DOI: 10.1371/journal.pone.0073503.]

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

Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

Intra-endolysosomal Ca(2+) release is required for endolysosomal membrane fusion with intracellular organelles. However, the molecular mechanisms for intra-endolysosomal Ca(2+) release and the downstream Ca(2+) targets involved in the fusion remain elusive. Previously, we demonstrated that endolysosomal P2X4 forms channels activated by luminal adenosine triphosphate in a pH-dependent manner. In this paper, we show that overexpression of P2X4, as well as increasing endolysosomal P2X4 activity by alkalinization of endolysosome lumen, promoted vacuole enlargement in cells and endolysosome fusion in a cell-free assay. These effects were prevented by inhibiting P2X4, expressing a dominant-negative P2X4 mutant, and disrupting the P2X4 gene. We further show that P2X4 and calmodulin (CaM) form a complex at endolysosomal membrane where P2X4 activation recruits CaM to promote fusion and vacuolation in a Ca(2+)-dependent fashion. Moreover, P2X4 activation-triggered fusion and vacuolation were suppressed by inhibiting CaM. Our data thus suggest a new molecular mechanism for endolysosomal membrane fusion involving P2X4-mediated endolysosomal Ca(2+) release and subsequent CaM activation

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by two-pore channel 2 inhibition

Two-pore channels (TPCs) are endo-lysosomal ion channels implicated in Ca2+ signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease however is unclear. Here we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators (Rab7, NAADP, PI(3,5)P2) and buffering local Ca2+ increases. NAADP-evoked Ca2+ signals were exaggerated in diseased cells. TPC2 is thus a potential druggable target within a pathogenic LRRK2 cascade that disrupts Ca2+-dependent trafficking in Parkinson disease

Investigation of proteins that interact with NAADP-Gated two-pore channels.

University of Minnesota Ph.D. dissertation. February 2012. Major: Pharmacology. Advisor: Dr. Jonathan Marchant. 1 computer file (PDF); ix, 84 pages.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.Moshier, Yaping Lin. (2012). Investigation of proteins that interact with NAADP-Gated two-pore channels.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/121764