Date of Award

May 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

David H Petering

Committee Members

Mahmun Hossain, Arsenio A Pacheco, Guilherme Indig, Nicholas Silvaggi

Keywords

Cellular Zinc Buffering, Intracellular Zinc Sensors, Intracellular Zinc Trafficking, Metallothionein, Proteome, Zinc Proteome

Abstract

Zinc is the second most abundant transition metal in living organisms. Typically, a eukaryotic cell contains approximately 3000 zinc binding proteins, in which zinc plays structural or catalytic roles. Recently, zinc has been reported to have signaling functions as a secondary messenger. Considering the diverse cellular functions of zinc, the trafficking pathways that help zinc, following its uptake into cytosol, find the target proteins and generate native Zn-Proteins are not well-understood. For past few decades, metallothionein, a zinc binding protein with large stability constants for Zn2+, has been thought of as a mediator in transferring zinc to apo-Proteins. However, gene knock-outs of metallothionein isoforms, reportedly, do not affect the birth and survival of metallothionein-null mice, implying that metallothionein is not absolutely required for zinc trafficking, but may have a supporting role in the process. Beyond metallothionein, zinc encounters numerous potential zinc binding ligands with varying stability constants, including the Proteome’s adventitious zinc binding sites and glutathione. In this research, we characterized the non-specific zinc binding sites of Proteome using a colorimetric zinc sensor and then investigated if Proteome, metallothionein and glutathione, alone or with the presence of others, can mediate the transfer of zinc to apo-Proteins to generate native Zn-

Proteins. For this, we have used Zn-carbonic anhydrase as a model Zn-Protein, and dansyl amide as a Zn-carbonic anhydrase sensor. The experimental results indicate that the mechanism of cellular zinc trafficking leading to the formation of native Zn-Proteins does not involve the straight-forward transfer of zinc from one particular cellular component or chaperone to apo-Proteins. Instead, trafficking occurs through a complex interconnected pathway that primarily includes proteomic non-specific zinc binding sites, metallothionein and glutathione.

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