Regulation of intracellular heme trafficking revealed by subcellular reporters

Abstract

Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditiselegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

Characterization of plasma labile heme in hemolytic conditions

“…However, these approaches are less sensitive, as compared to the ones we describe herein, and moreover do not allow discriminating what proportion of the labile heme becomes bioavailable, which is made possible in our study based on the use of a cellular-based heme reporter assay [31,62]. …”

“…Expression of HRP was confined to the Golgi with a targeting sequence from galactosyltransferase [12,31]. Control HEK293 cells were transiently transfected with the pEF5/FRT/V5-DEST vector [12,31]. Transfected cells were cultured (24 h) in DMEM (10% heme-depleted FBS) with or without succinylacetone (SA; 0.5 m m ) to block endogenous heme synthesis.…”

“…(C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [31]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D).…”

“…Here, we developed a panel of heme-specific sdAbs that, when used together with other approaches [31], allow to quantify and characterize labile heme in plasma, its rate of cellular internalization and cellular localization. We report that intravascular hemolysis in mice is associated with the accumulation of labile heme in plasma, which is bound to plasma molecules with an affinity higher than 10 −7 m .…”

Heme as a Target for Therapeutic Interventions

“…Other known extracellular plasma HBPs are α1-microglobulin (Allhorn et al, 2002) and α1-antitrypsin (Karnaukhova et al, 2012) (Table 1). Intracellular binding of heme by HBPs may not only protect against its potential pro-oxidant toxicity, but may be also involved in trafficking of heme between different cell compartments (Muller Eberhard and Nikkilä, 1989; Liem et al, 1994; Yuan et al, 2016). Thus, the specific functional roles of intracellular candidate HBPs in mammalians such as glutathione-S-transferases, heme-binding protein/ liver fatty-acid binding protein, heme-binding protein 23/peroxiredoxin 1, p22 heme binding protein and glyceraldehyde-3-phosphate dehydrogenase (Harvey and Beutler, 1982; Vincent and Muller Eberhard, 1985; Iwahara et al, 1995; Taketani et al, 1998; Chakravarti et al, 2010) need to be investigated in more detail (Table 1).…”