Date of Award
August 2016
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
David H. Petering
Committee Members
Graham Moran, David Frick, Arsenio Pacheco, Alexander Arnold
Keywords
Cellular Zinc Buffering, Newport Green, Zinc Fluorescent Sensors, Zinc Proteome, Zinc Trafficking, Zinpyr-1
Abstract
Zinc is an essential biological trace metal used in as many as 3000 Zn-proteins, about 10% of the eukaryotic proteome, as either a structural constituent or a catalytic cofactor. These proteins include the zinc fingers, the most prevalent transcription factors that bind a wide range of gene promoters and thus regulate gene expression. A eukaryotic cell contains several hundred micromolar of Zn2+- almost all of it is bound to specific Zn-proteins. Recently, Zn2+ has been reported to serve as a regulatory signal and a neurotransmitter, suggesting that there also exists a dynamic Zn2+ pool in cells. These findings led to the synthesis of a wide range of fluorescent sensors to image intracellular mobile Zn2+. Despite extensive knowledge about thousands of Zn-proteins, the Zn2+ trafficking pathway from its entry into the cytosol by transporters to the formation of Zn-proteins is not well understood. This present work has studied the role of proteome in cellular Zn2+ trafficking using fluorescent zinc sensors, including FluoZin-3, Zinquin (ZQ), TSQ, Newport Green (NPG) and Zinpyr-1 (ZP1). The titration of proteome pre-treated with FluoZin-3, a relatively high affinity Zn2+ sensor with the stability constant of 15 nM, with Zn2+ has revealed that proteome contains a significant number of high affinity, non-specific Zn2+ binding sites, with the stability constants on the order of 10-10 M. The discovery of these high affinity binding sites of proteome suggested that along with Zn-metallothionein, proteome too can serve as a possible intermediate along the way to the formation of native Zn-proteins. Moreover, this finding raises the question how the majority of the fluorescent zinc sensors with the stability constants ranging from micromolar to nanomolar image intracellular labile Zn2+ by circumventing the proteome’s high zinc buffering capacity. Interestingly, the thiol binding reagents, N-ethylmaleimide (NEM) and DTNB abolished these high affinity sites, revealing the involvement of proteomic sulfhydryl groups in these Zn2+ binding sites. The loss of proteome’s zinc buffering capacity upon sulfhydryl modification can explain how the sensors bind the dynamic Zn2+ pool by surpassing the proteome’s high Zn2+ binding affinity. For example, Zinquin, a high affinity sensor with Kd of 2 nM, could bind the mobile Zn2+ only when the proteome was significantly modified by the reaction under investigation, such as the liberation of proteomic Zn2+ by nitric oxide, which reacts with the sulfhydryl groups and thus reduces proteome’s buffering capacity. In case of unperturbed proteome, these sensors either are unable to compete for mobile Zn2+ with proteome’s high affinity Zn2+ binding sites or generate ternary adduct, Proteome•Zn-Sensor, with Zn2+ preferentially bound to proteome. Newport Green, for example, with its modest stability constant (Kd 10-5 – 10-6 M) cannot efficiently compete with proteomic ligands to image mobile Zn2+. It could not bind intracellular Zn2+ shuttled into LLC-PK1 cells using the ionophore, pyrithione. Moreover, when proteomic Zn2+ was liberated by the reaction with sulfhydryl binding reagents, NEM and diethylamine NONOate (DEA-NO), in the presence of Newport Green, insignificant amount of Zn-NPG was detected. By contrast, the higher affinity Zn2+ sensor (Kd 0.7 nM) than Newport Green, ZP1 formed ternary adduct Proteome•Zn-ZP1 with the dynamic Zn2+, where Zn2+ is adventitiously bound to proteome’s high affinity zinc binding sites. Besides with the mobile Zn2+, ZP1 also seems to react with the static distribution of cellular Zn2+ in specific Zn-proteins and generates ZP1-Zn-Proteome ternary adduct. Therefore, the effectiveness of the sensors to bind the cellular dynamic Zn2+ is a variable of proteome’s Zn2+ binding characteristics.
Recommended Citation
Karim, Mohammad Rezaul, "The Role of Proteome in Cellular Zn2+ Trafficking and in the Ability of the Fluorescent Zinc Sensors to Image Intracellular Zn2+" (2016). Theses and Dissertations. 1278.
https://dc.uwm.edu/etd/1278