Mucolipidosis type IV (MLIV) is a lysosomal storage disorder that mainly affects the brain, eyes, and stomach. The disease is caused by a loss of function mutation in the TRPML1 ion channel. We discovered abnormally high zinc levels in MLIV patient fibroblasts and brain tissues of the MLIV mouse model. Indeed, when MLIV fibroblasts are acutely exposed to exogenous zinc, marked accumulation of the ion is seen in lysosomes. These findings impact our understanding of MLIV pathology, because the brain contains a chelatable pool of zinc that is co-released with glutamate during normal neurotransmission, or pathological events. We identified transmembrane (TMEM)-163, a zinc-binding protein and putative transporter, as a novel interaction partner for TRPML1. Heterologous expression of TMEM163 shows that it localizes in the plasma membrane (PM), and that it partially co-localizes with TRPML1 in the lysosomes. Preliminary data show that TMEM163-overexpressing cells exposed to exogenous zinc result in significant increase of Fluozin-3 fluorescence and Metallothionein-1A expression, a marker of intracellular zinc overload. Also, our heterologous co-expression of TMEM163 and zinc transporter-4 (ZnT4), but not other ZnT proteins tested, revealed a synergistic increase of Fluozin-3 fluorescence. Meanwhile, cell surface biotinylation studies showed that the PM levels of wild-type (WT) and deletion mutant TMEM163 proteins become stabilized when co-expressed with WT TRPML1, while deletion mutants mis-localize without TRPML1 co-expression, implying that TRPML1 influences the trafficking of TMEM163. Thus, we hypothesize that TMEM163 is a transporter that mediates intracellular zinc homeostasis independent of, or in conjunction with other zinc transporters that could possibly contribute to MLIV disease. The first goal of this proposal is to investigate the putative zinc transporter function of TMEM163 by determining whether it is influx or efflux, and ATP- or pH-dependent using heterologously expressed cells and liposomes taken from cell membranes of TMEM163-mCherry overexpressing (OE) cells.
The second aim i s to identity specific amino acids within TMEM163 that are responsible for zinc transport, and determine if the synergistic function between TMEM163 and ZnT4 proteins is due to their physical interaction. Finally, the third goal will determine if TMEM163 trafficking in cells is mediated by TRPML1 upon TRPML1-OE or knock out, and upon exposure of cells with various drug inhibitors of trafficking processes. Ultimately, the data that will be gathered from this proposal would fill current gaps in our knowledge on the physiological or potentially detrimental role of TMEM163 with respect to MLIV etiology, and the mechanistic processes responsible for intracellular or lysosomal zinc accumulation leading to MLIV pathology.

Public Health Relevance

Zinc is a crucial trace metal in human biology, but is toxic at unusually high levels, especially in the brain. We discovered that zinc metabolism in Mucolipidosis type IV (MLIV) disease is abnormal, and this proposal will define the role of TMEM163 in cells in transporting zinc. Data from this study could provide a framework to devise a future therapy targeting abnormal zinc levels that would mitigate symptoms and prevent disease progression in children suffering from MLIV.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Academic Research Enhancement Awards (AREA) (R15)
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Special Emphasis Panel (ZRG1-MDCN-R (86)A)
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Morris, Jill A
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California State University Fullerton
Schools of Arts and Sciences
United States
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Chacon, Jonathan; Cuajungco, Math P (2018) Comparative De Novo Transcriptome Assembly of Notophthalmus viridescens RNA-seq Data using Two Commercial Software Programs. Calif J Health Promot 16:46-53
Cuajungco, Math P; Kiselyov, Kirill (2017) The mucolipin-1 (TRPML1) ion channel, transmembrane-163 (TMEM163) protein, and lysosomal zinc handling. Front Biosci (Landmark Ed) 22:1330-1343