Title:
Development of New Heme Sensors and Novel Insights into Heme Homeostasis
Development of New Heme Sensors and Novel Insights into Heme Homeostasis
Author(s)
Dominic, Iramofu Mark
Advisor(s)
Reddi, Amit R.
Editor(s)
Collections
Supplementary to
Permanent Link
Abstract
Heme is an essential iron-containing nutrient important for a myriad of biological processes and defects in heme homeostasis have been implicated in a variety of disorders. However, heme is potentially cytotoxic, suggesting that cells must have evolved mechanisms to limit the deleterious effects heme may have on membranes and proteins. While some heme processes are well characterized, many aspects of heme biology remain unclear. To enable the development of better medical solutions for heme-related conditions, there is a need to improve our understanding of heme homeostasis mechanisms. Previous work had developed genetically encoded heme sensors that have vastly increased our understanding of heme homeostasis across several different organisms, and to expand the ability of these tools to probe heme in multiple contexts, this work sought to target the heme sensors to the ER and diversify the color palette of heme sensors for multi–compartmental and –cellular imaging. Additionally, the novel phenomenon of intercellular heme transfer in Baker’s yeast was preliminarily characterized.
Attempts to target the prototype heme sensor (HS1) to the S. cerevisiae ER were unsuccessful due to an incompatibility between the integral fusion sensor design and yeast import and folding capacity. While ER heme sensors redesigned into a linear domain structure overcame fluorescence and localization challenges, they failed to recapitulate HS1 functionality. Interestingly, a humanized ER-HS1 was correctly localized and functional, validating its utility in expanding our understanding of heme biology, and highlighting species differences in heterologous protein expression.
The diversification of the HS1 color palette involved replacing the green fluorescence module with a cyan variant. The S. cerevisiae ECFP-HS1 functioned similarly to the prototype sensor, responding to titrations of labile heme levels across different strains. Importantly, the ECFP-HS1 signal was distinctly differentiable from HS1, allowing both constructs to be simultaneously utilized for multiplexing applications.
Finally, cell-cell communication and nutrient exchange in the context of heme was explored in S. cerevisiae. Heme-deficient strains were found to uptake heme from heme-replete donor strains in a manner that involved cell-cell contact. This finding has implications not only for our understanding of heme homeostasis, but also general nutrient regulation for yeast population control.
Taken together, this work has expanded the toolkit for probing heme trafficking and signaling in biological systems, as well as identified a novel process of heme regulation in Baker’s yeast.
Sponsor
Date Issued
2024-12-08
Extent
Resource Type
Text
Resource Subtype
Dissertation