Kukic, Ira
(2014)
Unclogging the zinc sink: examining the role of zinc trafficking in mucolipidosis type IV and lysosomes.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Zinc is an essential micronutrient that regulates many cellular processes. Deficits or excessive abundance in zinc concentration have catastrophic consequences for organisms and have been linked to many human diseases. Consequently, intracellular zinc levels are kept within narrow limits by an orchestrated network of zinc binding proteins and transporters. Within the cell, zinc accumulates in virtually all organelles, including lysosomes, which break down cellular waste materials. Lysosomes have recently become the focus of zinc regulation, as they have been shown to absorb zinc from the cytoplasm and contribute to zinc-induced toxicity. Additionally, recent evidence of zinc permeability of the lysosomal ion channel TRPML1, and the evidence of abnormal zinc levels in cells deficient in TRPML1, suggested a role for TRPML1 in zinc transport. Mutations in TRPML1 in humans result in a neurodegenerative lysosomal storage disorder called Mucolipidosis type IV (MLIV) characterized by psychomotor and mental retardation.
I have found that an siRNA-driven TRPML1 knockdown in HeLa cells leads to the buildup of enlarged cytoplasmic and lysosomal vesicles filled with zinc via live cell confocal imaging. Also, lysosomal enlargement and zinc buildup in TRPML1-deficient cells exposed to zinc are ameliorated by knocking down the lysosomal zinc importer ZnT4. This data provides important insights into the function of TRPML1 and the role of zinc in MLIV and other neurodegenerative diseases. Moreover, I have found that lysosomes, generally, play a cytoprotective role during exposure to extracellular zinc, which requires lysosomal acidification and exocytosis. Specifically, inhibition of lysosomal function with bafilomycin redistributed zinc pools to other organelles and increased cell death. Furthermore, inhibition of lysosomal exocytosis through knockdown of lysosomal SNARE proteins VAMP7 and SYT7 suppressed zinc secretion. VAMP7 knockdown increased apoptosis. Interestingly however, the overexpression of the lysosomal biogenesis transcription factor TFEB increased zinc secretion, suggesting that zinc secretion correlates with lysosomal function. Overall, these data underscore a role for TRPML1 and lysosomes in zinc metabolism and suggest that lysosomes form a “zinc sink” to dynamically buffer and counteract high zinc levels. This is a novel paradigm that implicates lysosomes in an entirely new function and identifies a completely new zinc detoxification pathway.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
29 May 2014 |
| Date Type: |
Publication |
| Defense Date: |
4 April 2014 |
| Approval Date: |
29 May 2014 |
| Submission Date: |
17 April 2014 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Number of Pages: |
212 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Zinc, Lysosome, TRPML1, Mucolipin-1, Mucolipidosis Type IV, Lysosomal exocytosis, secretion |
| Date Deposited: |
29 May 2014 16:13 |
| Last Modified: |
29 May 2019 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/21253 |
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