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Cellular zinc levels are modulated by TRPML1-TMEM163 interaction.

ABSTRACT: Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)-163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two-hybrid, tissue expression pattern, co-immunoprecipitation, mass spectrometry and confocal microscopy studies confirmed the physical association of TMEM163 with TRPML1. This interaction is disrupted when a part of TMEM163's N-terminus was deleted. Further studies to define the relevance of their interaction revealed that the plasma membrane (PM) levels of TMEM163 significantly decrease when TRPML1 is co-expressed in HEK-293 cells, while it mostly localizes within the PM when co-expressed with a mutant TRPML1 that distributes mostly in the PM. Meanwhile, co-expression of TMEM163 does not alter TRPML1 channel activity, but its expression levels in MLIV patient fibroblasts are reduced, which correlate with marked accumulation of zinc in lysosomes when these cells are acutely exposed to exogenous zinc (100??M). When TMEM163 is knocked down or when TMEM163 and TRPML1 are co-knocked down in HEK-293 cells treated overnight with 100 nm zinc, the cells have significantly higher intracellular zinc levels than untreated control. Overall, these findings suggest that TMEM163 and TRPML1 proteins play a critical role in cellular zinc homeostasis, and thus possibly explain a novel mechanism for the pathological overload of zinc in MLIV disease.

SUBMITTER: Cuajungco MP

PROVIDER: S-EPMC4205267 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Cellular zinc levels are modulated by TRPML1-TMEM163 interaction.

Cuajungco Math P MP Basilio Luigi C LC Silva Joshua J Hart Thomas T Tringali Jonathan J Chen Cheng-Chang CC Biel Martin M Grimm Christian C

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Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)-163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two-hybrid, tissue expression pattern, co-immunoprecipitation, mass spectrometry and confocal microscop ...[more]

PMID: 25130899

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Project description:Macroautophagy/autophagy is elevated to ensure the high demand for nutrients for the growth of cancer cells. Here we demonstrated that MCOLN1/TRPML1 is a pharmaceutical target of oncogenic autophagy in cancers such as pancreatic cancer, breast cancer, gastric cancer, malignant melanoma, and glioma. First, we showed that activating MCOLN1, by increasing expression of the channel or using the MCOLN1 agonists, ML-SA5 or MK6-83, arrests autophagic flux by perturbing fusion between autophagosomes and lysosomes. Second, we demonstrated that MCOLN1 regulates autophagy by mediating the release of zinc from the lysosome to the cytosol. Third, we uncovered that zinc influx through MCOLN1 blocks the interaction between STX17 (syntaxin 17) in the autophagosome and VAMP8 in the lysosome and thereby disrupting the fusion process that is determined by the two SNARE proteins. Furthermore, we demonstrated that zinc influx originating from the extracellular fluid arrests autophagy by the same mechanism as lysosomal zinc, confirming the fundamental function of zinc as a participant in membrane trafficking. Last, we revealed that activating MCOLN1 with the agonists, ML-SA5 or MK6-83, triggers cell death of a number of cancer cells by evoking autophagic arrest and subsequent apoptotic response and cell cycle arrest, with little or no effect observed on normal cells. Consistent with the in vitro results, administration of ML-SA5 in Patu 8988 t xenograft mice profoundly suppresses tumor growth and improves survival. These results establish that a lysosomal cation channel, MCOLN1, finely controls oncogenic autophagy in cancer by mediating zinc influx into the cytosol.Abbreviation: Abbreviations: 3-MA: 3-methyladenine; AA: amino acid; ATG12: autophagy related 12; Baf-A1: bafilomycin A1; BAPTA-am: 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; co-IP: coimmunoprecipitaion; CQ: chloroquine; DMEM: Dulbecco's Modified Eagle Medium; FBS: fetal bovine serum; GAPDH: glyceraldehyde- 3-phosphate dehydrogenase; HCQ: hydroxychloroquine; HEK: human embryonic kidney; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NC: negative control; NRK: normal rat kidney epithelial cells; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RPS6KB/S6K: ribosomal protein S6 kinase B; shRNA: short hairpin RNA; siRNA: short interfering RNA; SNARE: soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TPEN: N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; TTM: tetrathiomolybdate; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; Zn2+: zinc.

Project description:BackgroundSeed germination is a critical stage in plant life, and recent practices use nanomaterials for the improvement of plant seed germination indices. This study was conducted to assess the effect of laboratory prepared zinc oxide nanoparticles on the physiological and biochemical changes of lettuce seeds.MethodsLettuce seeds were soaked in a suspension of moderately polydisperse zinc oxide nanoparticles at two different concentrations (25 ppm or 50 ppm) and shaken for 3 h at 25 °C. Seeds treatment was followed subsequently by two to three days drying at ambient conditions. Treated seeds were stored for 3-4 weeks, at ambient conditions and then tested for germination in petri dishes. Germination was observed on daily basis and seedling length was measured. After imbibition and before the start of the visible germination, seeds were examined for topography and surface analysis using the scanning electron microscope and zinc uptake was measured by using the atomic absorption spectrometry and the energy dispersive X-ray. The pattern of mobilization of biomolecules was analyzed to detect any differences among different seed groups.ResultsThere was no loss of viability for the nanoparticles treated seeds. Indeed their germination was enhanced and their biomass increased. The activated performance of the nanoparticles imbibed seeds has been found to be correlated with an increased level of Zn inside lettuce seeds. The recorded measurements show a significant enhancement of seedling length. Interaction of zinc oxide nanoparticles with lettuce seeds mediates a variation in the biochemical processes. Changes detected in treated seeds were as following: reduced levels of the total carbohydrates (including simple saccharides and polysaccharides), higher capacity of protein synthesis, an elevated level of starch as well as an increased activity of antioxidant enzymes.Discussion and conclusionLettuce seeds primed with ZnO nanoparticles were found not only to maintain seed viability but even to exhibit a detectable level of germination enhancement compared to the control seeds. Overall, the promoted response of lettuce seeds during early stages of seed growth is encouraging for the application of ZnO NPs for seed priming for better germination indices.

Dataset Information

Cellular zinc levels are modulated by TRPML1-TMEM163 interaction.

Publications

Cellular zinc levels are modulated by TRPML1-TMEM163 interaction.

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