Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors

Abstract

Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.

Figure 1

Genetically encoded fluorescent heme sensors (A) CISDY and (B) HS1. Panel A was adapted with permission from ref . Copyright 2015 American Chemical Society. The molecular model of HS1 was generated using PyMol and is derived from the X-ray structures of mKATE [Protein Data Bank (PDB) entry 3BXB] and CG6 (PDB entry 3U8P).

Figure 2

Simulations of the dependence of the (A) fractional heme saturation of the sensor on labile heme concentrations and (B) observed labile heme concentration and fractional saturation of the heme sensor on sensor expression level. (A) The relationship between labile heme concentration and fractional heme saturation of the sensor is shown for sensors with heme dissociation constants of 2000 nM (black), 200 nM (red), 20 nM (yellow), 2 nM (green), and 0.2 nM (blue). Optimal heme sensing is achieved when the K_d of the heme sensor is similar to the concentration of labile heme. The simulation is based on the model depicted in eqs 1–3. (B) Dependence of the observed sensor fractional saturation (dashed lines) and the concentration of labile heme (solid lines) on sensor expression. The simulations were conducted using Hyperquad Simulation and Speciation HySS 2009, assuming a hypothetical competition between the heme sensor and a cellular heme buffer, present at 20 nM and having a heme K_D value of 20 nM, for 20 nM heme. Simulations are shown for three sensors with K_D values of 2 nM (orange and red), 20 nM (green and black), and 200 nM (light blue and dark blue).

Figure 3

Model of eukaryotic heme transport and trafficking. The final step of heme synthesis occurs in the mitochondrial matrix, and heme must be transported out of the mitochondria and incorporated into a multitude of hemoproteins found in different compartments. This process is likely mediated by heme chaperones and transporters. Proteins previously implicated in heme transport, trafficking, or buffering are identified, and trafficking pathways that are currently unknown are marked with question marks.