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

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

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

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

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

Oxidative Damage in MauG: Implications for the Control of High-Valent Iron Species and Radical Propagation Pathways

The di-heme enzyme MauG catalyzes the oxidative biosynthesis of a tryptophan tryptophylquinone (TTQ) cofactor on a precursor of the enzyme methylamine dehydrogenase (preMADH). Reaction of H(2)O(2) with the diferric form of MauG, or reaction of O(2) with diferrous MauG, forms the catalytic intermediate known as bis-Fe(IV), which acts as the key oxidant during turnover. The site of substrate oxidation is over 40 A from the high-spin heme iron where H(2)O(2) initially reacts, and catalysis relies on radical hopping through an interfacial residue, MauG Trp199. In the absence of preMADH, the bis-Fe(IV) intermediate is remarkably stable, but repeated exposure to H(2)O(2) results in suicide inactivation. Using mass spectrometry, we show that this process involves the oxidation of three Met residues (108, 114 and 116) near the high-spin heme through ancillary electron transfer pathways engaged in the absence of substrate. The mutation of a conserved Pro107 in the distal pocket of the high-spin heme results in a dramatic increase in oxidation of these Met residues. These results illustrate structural mechanisms by which MauG controls reaction with its high-valent heme cofactor and limits uncontrolled oxidation of protein residues and loss of catalytic activity. The conservation of Met residues near the high-spin heme among MauG homologues from different organisms suggests that eventual deactivation of MauG may function in a biological context. That is, methionine oxidation may represent a protective mechanism that prevents the generation of reactive oxygen species by MauG in the absence of preMADH