Heme iron accounts for the majority of the iron in the human body, and hemoglobin contains as much as 70% of the total iron content of a normal adult. Accordingly, iron and heme homeostasis plays very important roles in hemoglobin synthesis and erythropoiesis. Iron deficiency anemia is very common with an incidence of approximately one billion cases worldwide. The hallmark of microcytic hypochromic red blood cells (RBCs) in iron deficiency anemia has led to early studies in the 1950s that demonstrated the stimulation of hemoglobin synthesis by inorganic iron in immature erythroid cells. Iron is inserted into protoporphyrin IX to form heme in the last step of heme biosynthesis. Consequently, iron deficiency also leads to heme deficiency. Further studies in the 1960s showed that heme, not iron per se, was required for protein synthesis in reticulocytes since the iron-chelating agent, desferrioxamine, did not block the stimulatory effect of heme. Heme deficiency causes disaggregation of polysomes in reticulocytes. On addition of hemin, polysomes reform and protein synthesis is restored. Activation of heme-regulated inhibitor (HRI) in heme-deficienct reticulocyte lysates was discovered in 1969. Inhibition of protein synthesis under heme deficiency was associated with a marked decrease in the formation of 40S.eIF2.Met-tRNAiGTP (the 43S pre-initiation complex). Furthemore, addition of purified eIF2 both prevented and reversed the inhibition of protein synthesis in heme deficiency. In 1976 HRI was shown to be the heme-regulated eIF2α kinase that phosphorylates the α-subunit of eIF2 [reviewed in (Chen 2000)].
Depletion of HRI by polyclonal or monoclonal antibodies shows that HRI is principally responsible for the inhibition of protein synthesis during heme deficiency (Chen 2000). This was validated by targeted disruption of the HRI gene in mice (Han et al. 2001). Hri −/− reticulocytes have increased rates of protein synthesis and decreased levels of phosphorylated eIF2α (eIF2αP) in comparison to Hri +/+ reticulocytes. This increase in protein synthesis is accompanied by an increase in larger-sized polysomes in Hri −/− reticulocytes, demonstrating a higher rate of translational initiation. Furthermore, protein synthesis in Hri −/− reticulocytes is no longer dependent on heme. These results establish that HRI is essential for heme-regulated translation in reticulocytes (Han et al. 2001).
Regulation of HRI by Heme
Recombinant HRI expressed in Sf9 insect cells purifies as a hemoprotein with the three characteristic absorption peaks in the visible wavelength: the Soret band at 424 nm and the α- and β-bands around 550 nm. Furthermore, there are two distinct types of heme-binding sites per HRI homodimer. One type of binding site is nearly saturated with stably bound endogenous heme co-purified with HRI, while the other binding site is available to bind hemin reversibly (Fig. 2) (Chefalo et al. 1998). This second reversible heme-binding site is responsible for the downregulation of HRI activity by heme. The stoichiometry of two heme molecules per HRI monomer has been confirmed by direct measurement of heme chromophore through alkaline pyridine treatment of homogeneous heme-saturated HRI (Bauer et al. 2001; Chen 2007).
Conserved residues His75 and His120 were identified to be the proximal and distal heme ligand, respectively, in the N-terminus domain of HRI as illustrated in Fig. 3. Mutation of His75 and His120 individually to Ala in full-length HRI resulted in decreased sensitivity to heme inhibition, similar to N-terminally truncated HRI (Yen and Chen unpublished). This finding further underscores the importance of heme coordination in the N-terminus in the downregulation of HRI activity. Thus, HRI is a unique and novel hemoprotein with dual heme-binding domains. Furthermore, there is cooperation between the two heme-binding domains to achieve efficient heme regulation of HRI kinase activities. In this regard, heme has been shown to stabilize the binding of the N-terminus domain with N-terminally truncated HRI as well as to stabilize the binding of the N-terminus domain with the KI domain.
HRI contains two putative heme regulatory motifs (HRMs), Cys409/Pro410 and Cys550/Pro551, in mouse HRI, which are not present in the other three members of eIF2α kinases (Fig. 3). The significance of HRM motifs in heme regulation of HRI is still not clear. These two Cys residues have been mutated individually to Ser with no apparent effect on the heme responsiveness of HRI (Rafie-Kolpin et al. 2000). Additionally, these two Cys residues are not conserved in chicken, Xenopus, or fish HRI, indicating that they may not be important for the heme regulation of HRI. However, Igarashi et al. suggested that His120 and Cys409 are heme ligands in the full-length HRI (Igarashi et al. 2008).
Tissue Expression and Phylogenetic Conservation of HRI
HRI protein and mRNA are highly expressed in erythropoietic tissues of the bone marrow, spleen, and fetal liver. In addition, HRI expression also increases during erythroid differentiation. While there are reports of the expression of HRI in non-erythroid cells, it is important to note that HRI is expressed at two orders of magnitude higher levels in erythroid precursors as compared to macrophages (Liu et al. 2007). There are emerging evidence for the role of HRI in innate immunity and memory acquisition; however, it is beyond the scope of this review.
HRI homologues are present in vertebrates with amino acid sequence identity ranging from 45% in fish to 82% in humans. Importantly, the two heme ligands in the N-terminal heme-binding domain described above, His75 and His120, are conserved in all vertebrate HRI (Fig. 3). Interestingly, Schizosaccharomyces pombe not only has GCN2 but also has two additional eIF2α kinases, which have greater homology to HRI than to other eIF2α kinases. Although these two HRI-related eIF2α kinases can be regulated by heme in vitro, albeit with a higher Ki, these HRI-related kinases do not respond to iron/heme deficiency in vivo (Zhan et al. 2002) as mammalian HRI does (Han et al. 2001). They do, however, respond to oxidative and heat stress (Zhan et al. 2002) like mouse HRI in erythroid precursors (Lu et al. 2001). The pombe-like HRI-related kinases are present in Anopheles gambiae, Aedes aegypti, Tribolium castaneum, Bombyx mori, Branchiostoma floridae, Ciona intestinalis, and Nematostella vectensis. The N-terminus sequences of these HRI-related kinases are much less conserved than those of vertebrate HRI. Furthermore, His75 is no longer conserved, while His120 is replaced with Tyr in these HRI-related kinases (Fig. 3). There is no HRI in Saccharomyces cerevisiae, Caenorhabditis elegans, or Drosophila melanogaster.
The higher degree of conservation of vertebrate HRI from fish to human, the high-level expression of HRI in erythroid cells, and the high sensitivity of HRI to heme regulation support the notion that HRI evolved when diffusion alone could no longer supply the increasing demands for oxygen and when blood circulation was established wherein hemoglobin is carried in red blood cells. Vertebrate HRI retains its ability to respond to oxidative and heat stress similar to the pombe HRI-related kinase.
Role of HRI in Coordinating of Heme and Globin Synthesis and in Iron/Heme Deficiency Anemia
In the formation of stable α2β2 hemoglobins, it is important to keep the concentrations of globin chains and heme balanced. Globin chains misfold and precipitate in the absence of heme. Excess heme causes oxidative stress and is also cytotoxic. As described above, HRI is a hemoprotein with two distinct heme-binding domains, and as such HRI may sense heme availability and serve as a feedback inhibitor of globin synthesis to balance heme and globin synthesis as illustrated in Fig. 1 [reviewed in (Chen 2007)]. This notion was proven by studies using Hri −/− mice to reveal the physiological function of HRI (Han et al. 2001).
The importance of HRI in the pathophysiology of heme deficiency disorders is further demonstrated in mice with combined deficiencies of HRI and ferrochelatase (Fech) (Han et al. 2005). Since Fech (the last enzyme of heme biosynthesis) inserts iron into protoporphyrin IX to form heme, Fech-deficient mice are heme deficient. HRI is activated in Fech-deficient reticulocytes, providing the in vivo evidence that HRI is regulated directly by heme and not by iron (Han et al. 2005). Fech-deficient mice develop microcytic hypochromic anemia and erythropoietic protoporphyria. In HRI deficiency, however, these mice display more severe phenotypes and display macrocytic hyperchromic anemia. Furthermore, globin inclusions are observed in reticulocytes from HRI-deficient Fech-deficient animals. The morphologically and biochemically similar red cell abnormalities elicited by the absence of HRI both in iron and heme deficiencies further underscore the importance of HRI in inhibiting protein synthesis to avoid accumulation of excess heme-free globins in heme-deficient states.
Integrated Stress Response of eIF2αP Signaling in Primary Erythroblasts
Phosphorylation of eIF2α by eIF2α kinases elicits an integrated stress response (ISR) under various stress conditions and is conserved from yeast to humans (Ron 2007). In mammalian cells, four eIF2α kinases, HRI, PKR, GCN2, and PERK, are expressed in distinct tissues to combat different stresses. PKR responds to viral infection while GCN2 senses nutrient starvations. PERK is activated by endoplasmic reticulum (ER) stress, and HRI is inhibited by heme. All four eIF2α kinases respond to oxidative and environmental stresses (Chen 2000, 2007).
A major target gene activated by ATF4 is the transcription factor C/EBP homologous protein-10 (CHOP-10). CHOP is upregulated transcriptionally in a wide variety of cells upon many stresses. Induction of CHOP leads to expression of GADD34 (Fig. 5), which recruits eIF2αP for dephosphorylation by PPase1 (Fig. 1). This action of GADD34 in regenerating active eIF2 is necessary for the recovery of protein synthesis of stress-induced gene expression that occurs late in the stress response (reviewed in (Chen 2014)). Upon ER stress, ISR has been shown to upregulate expression of genes directly involved in redox homeostasis to mitigate oxidative stress (Harding et al. 2003). Increased ROS levels were observed in cells with impaired ISR signaling resulting from mutations in eIF2α phosphorylation (Scheuner et al. 2001) or from deletion of PERK (Harding et al. 2003).
HRI-eIF2αP-ATF4 Signaling Necessary for Erythroid Differentiation
Beyond regulation of globin translation, HRI is also necessary to reduce ineffective erythropoiesis during iron/heme deficiency and in β-thalassemia (Han et al. 2001, 2005). Recently, it has been shown that the ineffective erythropoiesis occurring in Hri −/− mice during iron deficiency is due primarily to the profound inhibition of erythroid differentiation at the basophilic erythroblast stage (Suragani et al. 2012). This is also observed in several other mouse models of stress erythropoiesis including β-thalassemia and in mice deficient of Rb or Stat5a/5b deficiency [reviewed in (Chen 2014)].
While Hri −/− fetal liver (FL) displayed a mild defect in erythroid differentiation in vivo under normal iron-sufficient conditions (Liu et al. 2008), Hri −/− FL erythroid progenitors showed a significant inhibition of erythroid differentiation at the basophilic erythroblast stage when cultured and differentiated ex vivo (Suragani et al. 2012), recapitulating the inhibition of erythroid differentiation in vivo during iron deficiency (Liu et al. 2008; Suragani et al. 2012). The HRI-eIF2αP-ATF4 pathway is also activated during ex vivo differentiation of erythroid precursors and during erythroid differentiation of mouse erythroleukemic (MEL) cells. Furthermore, knockdown of ATF4 in MEL cells resulted in inhibition of erythroid differentiation (Suragani et al. 2012). Thus, the HRI-ATF4 signaling pathway may be necessary for inducing transcription of genes required for erythropoiesis starting at the basophilic erythroblast stage. As summarized in Fig. 7, HRI not only inhibits globin translation in nucleated erythroblasts but also increases ATF4 translation to mitigate oxidative stress and to promote erythroid differentiation. At the enucleated reticulocyte stage, the role of HRI is to regulate globin translation to prevent excessive globin synthesis, which is cytotoxic and increases oxidative stress. Both of these functions of HRI are necessary for optimal erythroid maturation to prevent anemia.
HRI is a key regulator of protein synthesis and differentiation in the erythroid lineage under stress conditions of iron deficiency and oxidative stress as well as in disease states of thalassemia and erythropoietic protoporphyria. It senses intracellular concentrations to coordinate globin translation with the heme available for the production of hemoglobin. Additionally, translational upregulation of ATF4 mRNA by HRI-eIF2αP signaling is important not only for mitigating oxidative stress but also for promoting erythroid differentiation. However, ATF4 mRNA may not be the only target of HRI-eIF2αP signaling during erythropoiesis. Ribosome profiling, which assesses ribosome occupancies in translating mRNAs in vivo genome-wide, will be instrumental in uncovering additional translational initiations at uORFs. Defining these translationally regulated and long-sought target uORF containing mRNAs of eIF2αP signaling in Wt and Hri −/− erythroid precursors will reveal novel protein components in regulating erythropoiesis. Further studies on the molecular mechanisms of HRI-eIF2αP-ATF4 signaling in the regulation of erythroid differentiation in iron and heme deficiencies may be illuminated by using mutant mice defective in this signaling pathway such as Atf4 −/− and the erythroid-specific Ala51 substitution of eIF2α in comparison with Hri −/− mice. Finally, HRI-eIF2αP signaling may be exploited for a novel pharmaceutical therapy for hemoglobinopathies.
- Chen J-J. Heme-regulated eIF-2α kinase. In: Sonenberg N, JWB H, Mathews MB, editors. Translational control of gene expression. Cold Springs Harbor: Cold Spring Harbor Laboratory Press; 2000. p. 529–46.Google Scholar
- Igarashi J, Murase M, Iizuka A, Pichierri F, Martinkova M, Shimizu T. Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins. J Biol Chem. 2008;283:18782–91.PubMedCrossRefGoogle Scholar
- Ron D, Harding HP. In: Mathews NSM, Hershey JWB, editors. eIF2α phosphorylation in cellular stress responses and disease. In translational control in biology and medicine. Cols Spring Harbor: Cols Spring Harbor Laboratory Press; 2007.Google Scholar
- Zhan K, Vattem KM, Bauer BN, Dever TE, Chen JJ, Wek RC. Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for fesistance to environmental stresses. Mol Cell Biol. 2002;22:7134–46.PubMedPubMedCentralCrossRefGoogle Scholar