Update on heme biosynthesis, tissue-specific regulation, heme transport, relation to iron metabolism and cellular energy

Abstract

Heme is a primordial macrocycle upon which most aerobic life on Earth depends. It is essential to the survival and health of nearly all cells, functioning as a prosthetic group for oxygen-carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Heme is essential for the function of numerous hemoproteins and has numerous other roles in the biochemistry of life. In mammals, heme is synthesised from glycine, succinyl-CoA, and ferrous iron in a series of eight steps. The first and normally rate-controlling step is catalysed by 5-aminolevulinate synthase (ALAS), which has two forms: ALAS1 is the housekeeping form with highly variable expression, depending upon the supply of the end-product heme, which acts to repress its activity; ALAS2 is the erythroid form, which is regulated chiefly by the adequacy of iron for erythroid haemoglobin synthesis. Abnormalities in the several enzymes of the heme synthetic pathway, most of which are inherited partial enzyme deficiencies, give rise to rare diseases called porphyrias. The existence and role of heme importers and exporters in mammals have been debated. Recent evidence established the presence of heme transporters. Such transporters are important for the transfer of heme from mitochondria, where the penultimate and ultimate steps of heme synthesis occur, and for the transfer of heme from cytoplasm to other cellular organelles. Several chaperones of heme and iron are known and important for cell health. Heme and iron, although promoters of oxidative stress and potentially toxic, are essential cofactors for cellular energy production and oxygenation.

1 |. MAMMALIAN HEME METABOLISM AND ITS REGULATION

Heme (iron protoporphyrin) is a primordial macrocycle upon which nearly all aerobic life on Earth depends. It is essential to the survival and health of nearly all cells, functioning chiefly as a prosthetic group for oxygen-carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Heme is essential for the function of numerous hemoproteins and, as described further below, has numerous other and still expanding roles in the biochemistry of life.

1.1 |. Heme biosynthesis and breakdown

In α-proteobacteria, fungi, and most metazoans, heme is synthesised from the building blocks of succinyl-CoA and glycine and includes eight enzymes (Figure 1). Diseases associated with abnormalities, mostly inherited, of the genes of the enzymes of heme synthesis are described in greater detail in other contributions of this special issue of Liver International. The pathway’s first and normally rate-controlling enzyme is 5-aminolevulinate (ALA) synthase (EC 2.3.1.37). This enzyme, which functions as a homodimer, carries out the following reaction: succinyl-CoA + glycine → ALA + CoA + CO₂. In eukaryotes, ALA synthase functions only within the mitochondria, perhaps related to the supply of succinyl-CoA. It forms molecular associations with two enzymes that synthesize succinyl-CoA, namely, succinyl-CoA synthetase and alpha-ketoglutarate dehydrogenase, the latter an essential enzyme in the tricarboxylic acid (TCA) cycle.²

FIGURE 1.

Overview of heme synthesis and breakdown showing pathway intermediates and end-product regulation by heme. The eight steps of heme synthesis [left column] are shown with the enzymes that catalyse the steps [middle column] and the diseases associated with abnormalities in the enzymes [right column]. [copyright 2017, Mass Medical Society; from Bissell et al.,¹ with permission of the authors and publisher].

An essential co-factor for ALA synthase is pyridoxal phosphate (PLP), which binds to a key lysine residue at the enzyme’s active center. Detailed studies of the reaction cycle have established that the release of the product ALA from the enzyme is the overall rate-controlling step.² As shown in Table 1, ALA synthase is notable for its high Km of glycine, suggesting that restriction of glycine supply to the enzyme might restrict its overall activity. However, competition for succinyl-CoA for the TCA cycle may also affect ALA synthase activity under some cellular energetics conditions.

TABLE 1.

Kinetic parameters for enzymes of heme biosynthesis and breakdown in livers of normal adult humans and other species.

	V max		K m
Enzyme	(nmol ALA Equiv/g Wet Liver/Hour)		(μM)
Heme synthesis
5-Aminolevulinic acid (ALA) synthase-1
Human	10–44	{	Glycine, 5000–19 000
Rat	20–60		Succinyl CoA, 6–120
Mouse	40–84		Pyridoxal PO4, 1–10
5-Aminolevulinic acid (ALA) dehydratase
Human	1660–3040	{
Rat	1400,3 3600		100–400
Mouse	3000–9600
Porphobilinogen deaminase [aka HMBS]
Human	17–24		6–14a
Rat	151
Mouse	70–180		37
Uroporphyrinogen decarboxylase
Human	572–2520
Rat	52, 920 ± 86
Mouse	180–240		1–5
Coproporphyrinogen oxidase
Human	520–2458		0.91
Rat	690, 288		48
Mouse	1200		30 (for ox liver)
Protoporphyrinogen oxidase
Rat	3270, 3920		11
Ferrochelatase
Human	520, 2800
Rat	2630, 4020, 11 400		Fe2+, 22
			Protoporphyrin, 0.8, 28.5
Heme breakdown	(nmol heme Equiv/g/h)
Heme oxygenase-1
Human
Rat	406 (total activity)a		0.24
Heme oxygenase-2
Human
Rat	406 (total activity)a		0.67
Biliverdin reductase
Human	2358 (total activity)b		BV, 0.8–1.0b
Rat	8494 (total activity)c		BV, 3.0c

Note: For the enzymes of heme synthesis, enzymatic activities are expressed as nmol of 5-aminolevulinic acid (ALA) equivalents produced or utilised per hour by 1 g wet liver. Some values were calculated assuming 150 mg protein/g wet liver or 40 mg mitochondrial protein/g wet liver. For enzymes of heme breakdown, Vmax values are expressed as nmol heme equiv/g wet liver/h. Adapted from Bonkovsky⁴ and Bonkovsky HL,¹⁵⁹ Elbirt KK. Chapter 41—heme oxygenase: its regulation and role. In: Cutler RG, Rodriguez H, eds. Oxidative Stress and Aging: Advances in Basic Science, Diagnostics, and Intervention, World Scientific Publ. 2002:699–706. Used by permission of the authors and publishers.

^aData from Vreman and Stevenson.¹⁵⁶ Anal Biochem. 1988;168:31–38.

^bData from Yamaguchi et al.¹⁵⁷ J Biol Chem. 1994;269:24343–24348.

^cData from Kutty et al.¹⁵⁸ J Biol Chem. 1981;256:3956–3962.

The second step in heme synthesis is condensation of two molecules of ALA to form the monopyrrole porphobilinogen (PBG). This reaction is carried out by the enzyme PBG synthase, also known as ALA dehydratase. It functions as an octamer, and its Vmax is very high, the highest of the enzymes of heme synthesis (Table 1). PBG synthase requires zinc for activity, and it is strongly inhibited by lead and by 4,6-dioxohepatanoic acid, an intermediate that accumulates in hereditary tyrosinemia type-1. PBG synthase is an iron–sulfur (Fe4S4) cluster protein.⁵ Neurological features of acute hepatic porphyrias resemble those seen in acute lead poisoning and hereditary tyrosinemia type 1, implicating high ALA levels as a likely key pathogenic factor in these disorders.^1,6 The third enzyme in the heme synthetic pathway is hydroxymethylbilane (HMB) synthase, or PBG deaminase. It carries out the polymerisation of four molecules of PBG to form the tetrapyrrole HMB, which serves as the key building block not only for heme but also for chlorophyll and cobalamin. As shown in Table 1, its Vmax in mammalian liver is very low, nearly as low as that of ALA synthase. Thus, under conditions of up-regulation of ALA synthase, the activity of HMBS may become the secondarily rate-limiting step of heme synthesis, with marked accumulations of ALA and PBG, as occurs in AHP.

The fourth enzyme in mammalian heme synthesis is uroporphyrinogen 3 synthase (URO3S), or uroporphyrinogen 3 co-synthase. It carries out the inversion of the D ring of uroporphyrinogen I to form the 3 isomers. Inherited or acquired deficiency of URO3S gives rise to a variably severe cutaneous porphyria, CEP. Normally, the high activity of URO3S assures that little uroporphyrinogen 1 forms.

The 5th enzyme of heme biosynthesis is uroporphyrinogen decarboxylase (UROD), which functions as a homodimer without the need for cofactors and carries out the stepwise decarboxylation of the acetic acid beta-substituent side chains of uroporphyrinogen 3 to coproporphyrinogen 3. The order of these reactions among the rings of uroporphyrinogen 3 is normally D, A, B, and C. UROD is also able to decarboxylate uroporphyrinogen-1 to its corresponding hepta-, hexa-, penta-, and tetra-carboxylate congeners. This enzyme is subject to inhibition by factors that increase oxidative stress in hepatocytes, such as excess iron, alcohol, chronic hepatitis C, and estrogens, which are among the risk factors for PCT, which in much of the world is the most common form of clinically manifested porphyria^7,8 Uroporphomethenes, derived from uroporphyrinogens, are inhibitors of UROD important in the pathogenesis of PCT.⁹ About 25% of patients with PCT also have a genetically determined partial (~50%) deficiency of UROD activity, which is an additional risk factor for PCT, but which is insufficient to lead to the 75% or greater deficiency in UROD activity required for clinical disease. Severe homozygous or compound heterozygous deficiency of UROD gives rise to a severe photo-mutilating disease hepato-erythropoietic porphyria (HEP), with onset in early childhood.

In the next step of heme synthesis, the coproporphyrinogen 3, but not the coproporphyrinogen 1, formed by UROD, is oxidised to protoporphyrin 9 (PP), by the enzyme coproporphyrinogen oxidase (CPOX). This enzyme is in the mitochondria. Partial deficiency of CPOX, usually ~50% of normal, gives rise to the disease hereditary coproporphyria (HCP), the mildest of the AHPs.

In mitochondria, protoporphyrinogen 9 is next oxidised to PP by the enzyme protoporphyrinogen 9 oxidase (PPOX). Partial deficiency of this enzyme gives rise to variegate porphyria (VP), an AHP that mainly presents with cutaneous manifestations of bullae and blisters on sun-exposed skin but may also cause acute and severe neuro-visceral disease attacks.

Ferrochelatase (FECH), or heme synthase, inserts ferrous iron into the PP ring to form heme. Critical deficiency (>75%) of activity of FECH causes accumulation of PP in nascent RBCs and gives rise to the disease erythropoietic protoporphyria (EPP), which typically presents as acute and severe photosensitivity in early infancy. In EPP, there is marked over-production of PP, which, due to its hydrophobicity, is not excreted into the urine, unlike more highly carboxylated porphyrinogens or porphyrins. Rather, excess PP must be taken up by hepatocytes and secreted into the bile to be removed from the body. Excess PP is toxic to hepatocytes and cholangiocytes and may give rise to liver disease (protoporphyric hepatopathy).

1.2 |. Breakdown of heme

Although heme, especially as an essential co-factor in cytochromes P450 (CYP), may be broken down by some drugs and chemicals, which are suicide substrates of CYPs, the physiologic route of heme catabolism is by way of the enzyme heme oxygenase (HMOX), the first and rate-controlling enzyme for heme degradation. HMOX degrades heme to equimolar amounts of biliverdin 9-alpha, carbon monoxide (CO), and iron.^10–14 It may play a role in the regulation of ALAS1 by its effects on the regulatory heme pool.^15–19 The transcription factor BACH1 serves as a repressor of HMOX1 gene expression. At least a portion of heme-dependent up-regulation of HMOX1 is due to the BACH1/heme interaction.^20,21 Some microRNAs (miRNAs), with perfect seed sequence binding sites on BACH1 mRNA, directly lead to down-regulation of BACH1 mRNA and protein and thus up-regulation of HMOX1 gene expression.²² In addition, heme and other selected metalloporphyrins (MePNs) and microRNAs (miRNAs) influence the expression of BACH1 and/or stability of the BACH1 protein.^23–25

1.3 |. Diverse functions of heme

Heme is well known to be an essential cofactor for proteins such as haemoglobin, myoglobin, and neuroglobin that transport oxygen to tissues and the removal and excretion of carbon dioxide from tissues (Figure 2). It is also an essential co-factor for many oxidoreductases, such as mitochondrial cytochromes and cytochromes b5 and P-450, that play essential roles in the metabolism of an astonishingly diverse array of endogenous and exogenous compounds, drugs, and other xenobiotics.^6,26 Unsurprisingly, heme has been found to have diverse roles.³ Heme plays a key role in miRNA processing by binding and interacting with DiGeorge critical protein 8 (DGCR8).^27,28 Heme regulates numerous genes besides ALA synthase-1 and BACH1, including Rev-Erb-α and other genes involved in circadian rhythms.²⁹ Heme also modulates ion channel activities,^30–32 activates sensors,^33–35 promotes adipogenesis,^36,37 inflammation,^38–40 and exerts diverse effects on blood coagulation.^41–43 There can be little doubt that, as time goes by, more functions and roles of heme in additional aspects of metabolism will be unveiled.

FIGURE 2.

Examples of the diverse functions of heme. Biochemical systems and targets affected by heme with example proteins (in brackets), whose functions are modulated upon heme binding. ALAS1, 5-aminolevulinic acid synthase 1; Bach-1, BTB domain and CNC homologue 1; Bach-2, BTB domain and CNC homologue 2; C1q, complement component 1q; C3, complement component 3; CD74, cluster of differentiation 74; CLEC2, C-type lectin-like type II transmembrane receptor; DGCR8, DiGeorge critical region 8; FVIII, anti-hemophilic factor; IL-36, interleukin-36; Kv10.1, potassium channel protein 10.1; MD2, myeloid differentiation factor 2; Nav1.5, sodium channel protein type 5; NMDA, N-methyl-D-aspartate; RAGE, receptor for advanced glycation end products; Rev-erbB, nuclear receptor subfamily 1 group D; TLR4, Toll-like receptor 4; TNF, tumour necrosis factor.

1.4 |. Role and regulation of key enzymes in heme metabolism

1.4.1 |. ALA synthase and its regulation

ALA synthase (ALAS) is the first and normally rate-controlling enzyme of hepatic heme synthesis. In mammals, there are two forms of ALAS, ALAS1 and ALAS2. The two forms arose from gene duplication in ancestral vertebrates ~550 million years ago.² Selected features of ALAS1 and 2 are summarised in Table 2. The two isoforms are 66% similar and 60% identical in their amino acid compositions. Both function as homodimers with PLP as the essential cofactor. ALAS1 is the ubiquitous housekeeping form of the enzyme, found in all cells that contain mitochondria and the capacity for heme synthesis. The gene for ALAS1 is located on the short arm of chromosome 3. It is synthesised as pre-ALAS1, with an N-terminal ‘leader sequence’ that is cleaved during the transfer of the enzyme into the mitochondria. Its promoter and enhancer regions contain several consensus binding sites for nuclear receptors, including CAR, PPAR, PXR, and PGC1α. The human gene contains two non-coding exons, 1A and 1B, in its 5’-UTR, unlike the rat gene, which contains only 1 and the chick gene, which has none. The human gene is transcribed into 2 mRNAs, a major one, accounting for ~90% of the steady state concentration, in which exon1 is omitted, and a minor one that contains both exons 1A and 1B.⁴⁴ Of interest, the minor form, unlike the major, is resistant to heme-dependent decay, which suggests that the translation of mRNA into protein may be required for destabilisation in response to heme.⁴⁵

TABLE 2.

Aspects of human ALA synthase-1 and ALA synthase-2, the first and rate-controlling enzymes of heme synthesis.

Variable	ALA synthase-1	ALA synthase-2
Other names	Housekeeping, ubiquitous	Erythroid
Chromosomal location of gene	3 p.21	X p.11.21
Molecular wgt (Da)	70 581	64 633
# of amino acids	640	587
Factors that regulate gene transcription	Heme; glucose/insulin—down-regulate Corticosteroids/adrenal cortical function—permissive effect	Erythroid transcription factors—ELK/Sp1, GATA, HIF-1, NF-E2—up-regulate
Factors that regulate translation	Heme—decreases stability of mRNA	Supply of iron—via IRPs interacting with 5’-IRE's—increases translation
Factors that regulate mitochondrial uptake	Heme—via CP Heme Regulatory Motifs
Factors that regulate stability of the enzyme in mitochondria	Heme—increases LonP, mitochondrial protease that breakdowns the mature enzyme Heme activates Clpx, another protease—said to increase activity thru PLP, but also to increase breakdown of mature enzyme

It is unknown whether givosiran, the siRNA that down-regulates hepatic ALAS1 mRNA and decreases recurrent acute attacks of human porphyria,^46–49 leads to breakdown of both mRNA isoforms. It is striking that givosiran, even at higher doses, has not led to a deficiency of the mRNA in patients with AIP, but only to down-regulation to the normal, physiologic level of the mRNA.

ALAS 1 is markedly downregulated by heme^26,50 via blockade of gene transcription, mRNA breakdown, inhibition of mitochondrial uptake of the protein, and increased proteolytic breakdown of the mature mitochondrial form of ALAS1. Thus, the size of a postulated ‘regulatory heme pool’ in hepatocytes influences activity of ALAS1.⁵¹ This repressive effect may occur via a heme-mediated up-regulation of the early growth factor response 1 (EGR1) transcription factor, the assembly of which with its corepressors NAB1/2 onto heme responsive elements located in the proximal region of the ALAS1 gene, suppresses its expression.⁵² Another mechanism involves heme’s regulation of Rev-erb-α, which decreases transcription of PPAR1α.⁵³ Heme also increases mRNA degradation rates^16,54 by binding to heme responsive CP motifs and interfering with translocation of pre-ALAS into mitochondria.^16,55 In addition, heme increases the breakdown of mature mitochondrial ALAS1 by the ATP-dependent Lon peptidase 1⁵⁶ and ClpX⁵⁷ of mitochondria. Earlier studies in yeast had indicated that ClpX can increase ALAS activity by enhancing binding of its cofactor PLP,⁵⁸ but, in mammals at least, the more important effect of ClpX is likely on breakdown of ALAS1.

Several nuclear receptors (e.g. CAR, RAR, PXR) have been shown to bind to consensus binding sites of the ALAS1 promoter-enhancer regions.⁵⁹ Activity of hepatic HMBS, the third enzyme of the heme biosynthetic pathway, is normally only slightly greater than that of ALAS1. Thus, if ALAS1 activity is induced, HMBS becomes secondarily rate-limiting for heme synthesis. A 50% decrease in activity of hepatic HMBS is the hallmark of acute intermittent porphyria (AIP) the most severe of the acute porphyrias. Little is known about regulation of HMBS activity, although its substrate has been found to stabilize the enzyme.⁶⁰

ALAS1 is also down-regulated by glucose and other readily metabolised carbohydrates, a phenomenon called the ‘glucose effect.^4,6,61–63 One molecular mechanism of this effect is through modulation of PGC1-α by glucose and insulin.⁶⁴ Insulin has been shown to decrease ALAS1 in rat hepatocytes and human hepatocellular carcinoma cells,⁶⁵ although in other systems, insulin, thyroid, and adrenal cortical hormones have been found to have permissive or synergistic effects on ALAS1 activity.⁶⁶ The repressive effects of carbohydrates and insulin on ALAS1 continue to be important aspects of the management of acute porphyric attacks.^4,6

1.4.2 |. ALA synthase 2 and its regulation

ALAS2 is primarily expressed in developing red blood cell precursors, especially in erythroblasts. Regulation of ALAS2 is very different that of ALAS1 (Table 2). ALAS2 transcription is up-regulated by several erythroid transcription factors, including ELK/Sp1, GATA1,⁶⁷ HIF-1 and NF-E2.^68,69 While there may be some effects of heme on expression of the ALAS2 expression,⁷⁰ the major effect is mediated by the supply of iron. Thus, under conditions of iron deficiency, activity of ALAS2 is repressed, such that PP production will also be restricted, avoiding its toxic overproduction. Control of expression of ALAS2 mRNA by iron is mediated by IRP 1 and 2, which in the presence of sufficient iron, are released from IRE on the 5’ UTR of ALAS2, allowing for translation to proceed. Other mRNA’s regulated by IRP-IRE interactions include those of DMT1, FPN, ferritin H and L chains, and HIF2α.^71,72

Mutations that decrease levels/activities of ALAS2 give rise to X-linked sideroblastic anemias with iron overload in the mitochondria of erythroblasts. The severity of the anaemia is variable; some patients respond favourably to supplementation with pyridoxine or PLP, its active form. In contrast, deletions in exon 11 of ALAS2 lead to constitutive increases in activity of the enzyme and to over production of PP, the condition known as XLP. Patients with XLP exhibit increased levels of ZnPP in RBCs and respond favourably to iron replacement when they are iron-deficient, with increased production of heme and decreased accumulation of PP.

2 |. HEME TRANSPORT

The existence and role of heme importers and exporters in mammals have been debated.⁷³ Recent evidence, however, established the presence of heme transporters raising the possibility that cells and organs could share their heme to maintain cellular heme levels and homeostasis.

2.1 |. Heme import

The cytotoxic and hydrophobic nature of heme prevents it from spontaneously diffusing within the aqueous cytosol and across cell membranes.⁷⁴ C. elegans, a heme auxotroph, entirely relies on dietary heme for its survival. Exploiting the power of C. elegans, laid the groundwork for identification of more than 200 HRGs.⁷⁵ Intestinal heme absorption is mediated by HRG1 homologues, which have vertebrate orthologs.⁷⁶ Mammalian HRG1 is localised on the endolysosomal membranes and phago-lysosomal membranes of RES macrophages where it functions to import heme into the cytosol, thus contributing to iron recycling from senescent red blood cells (Figure 3).^77,78 The ablation of HRG1 in mice leads to hyperaccumulation of heme in lysosomes, leading to hemozoin crystal formation⁷⁹ typically found in blood-feeding parasites such as Plasmodium falciparum, resulting in darkened liver, spleen, and bone marrow.

FIGURE 3.

Model of major components of the heme trafficking pathway. Heme is either synthesised by the mitochondria or imported by transporters (in blue). A cell can also import heme through heme importers such as HRG1, FLVCR2, and endocytosis of senescent RBCs. Furthermore, different complexes are endocytosed to release heme, such as hemopexin/heme, haptoglobin/haemoglobin, and TFR1/hemealbumin. Heme is then transferred by HRG-1 from the endo-lysosome, and by FLVCR1b from the mitochondria to the cytoplasm. Several plasma membrane transporters (in green), FLVCR1a, MRP5, and ABCG2 export heme from the cell. A labile heme pool (LH) is envisioned to facilitate heme trafficking within the cell. In the nucleus, heme binds transcription factors to regulate transcription of target genes. ABCG2, ATP-binding cassette subfamily G member 2; CD, cluster of differentiation; FLVCR, feline leukaemia virus subgroup C receptor; GAPDG, glyceraldehyde-3- phosphate dehydrogenase; HBP[s], heme binding protein[s]; HRG, heme responsive gene; LH, labile heme; MRP, multidrug resistance protein; PGMRC[s], progesterone membrane receptor component[s]; RBC, red blood cell; TFR1, transferrin receptor 1.

How heme is transported by HRG1 is a question that remains to be explicated.⁸⁰ The mechanisms regulating HRG1 expression are poorly understood. Studies have identified heme-dependent transcriptional repressor BACH1 binding sites in HRG1 (Slc48a1), Hmox1, and Fpn1 promoters. However, HRG1 expression was not affected by BACH1 knock-down in HEK cells.⁸¹

Apart from HRG1, FLVCR2 (MFSD7C) was proposed as a plasma membrane heme importer.⁸² In humans, abnormalities in this protein are associated with Fowler syndrome, a brain proliferative vasculopathy.^82,83 Although FLVCR2 was proposed as a calcium-chelate transporter, Duffy et al. showed an increase in heme uptake and sensitivity to heme toxicity in cells expressing FLVCR2, as well as a loss of heme import while FLVCR2 is silenced.⁸² Recently, FLVCR2 was shown also to transport choline.

More recently, Tfr1 has been proposed to bind and endocytose albumin-heme in vitro. After the endocytosis of the complex, the labile iron pool increases, and this mechanism is dependent on HMOX1.⁸⁴

2.2 |. Heme export

FLVCR1 was the first heme exporter discovered and described as a plasma membrane transporter essential for erythropoiesis. Later, two isoforms of FLVCR1 were described, FLVCR1a and FLVCR1b, localising to the plasma membrane and the mitochondria, respectively.^85–87 More studies are required to determine if FLVCR1b localises on the inner or outer mitochondrial membrane.⁸⁸ FLVCR1a corresponds to the full-length form with 12 TMD, whereas FLVCR1b is a truncated form of the protein with only 6 TMDs due to an alternate start site in the first intron.⁸⁵ FLVCR1a transports heme from the cytosol, whereas FLVCR1b is implicated in heme export from the mitochondria after heme biosynthesis (Figure 3).⁸⁵ FLVCR1 is expressed in several tissues, including bone marrow, liver, and small intestine.⁸⁹ FLVCR1 null mice lacking both 1a and 1b die in utero at mid-gestation due to an erythropoiesis blockade.⁸⁶ Which of the two isoforms is required for erythropoiesis is debated; one study showed that FLVCR1a is essential during erythropoiesis to protect erythroid progenitors from heme toxicity, whereas another showed that FLVCR1b and not 1a is required for erythropoiesis. The role for FLVCR1a relies on the observation that heme accumulates within the cytosol due to loss of FLVCR1a resulting in apoptosis and aborted erythropoiesis.

Other studies suggest that FLVCR1 and 2 act as choline transporters.^90–92 Kenny et al. identified FLVCR1 as a genetic determinant of phosphocholine and phosphatidylcholine levels in human plasma. Furthermore, loss of FLVCR1 in HEK293T and HeLa cells impairs choline metabolism. Using radiolabeled choline uptake assays, they demonstrated that FLVCR1 transports choline into mammalian cells. FLVCR1^−/− mouse embryos display defects in choline metabolism and mitochondrial structure.

MRP-5/ABCC5 was discovered in C. elegans and demonstrated as a heme exporter in worms, yeast, zebrafish, and mammalian cell lines. It is expressed on the basolateral membrane of the worm intestine and on the plasma membrane, Golgi complex, and recycling endosomes in zebrafish.⁹³ MRP5 belongs to the ABC transporter superfamily, which is implicated in cancer drug resistance. MRP5 is expressed nearly ubiquitously in mammals, yet loss of MRP5 has no overt phenotype in mice.⁹⁴ Its deficiency in worms leads to embryonic lethality due to loss of heme export and consequently to heme deficiency in extra-intestinal tissue.⁹³ In zebrafish, MRP5 knock-down leads to severe anaemia.⁹³ One hypothesis for the difference between the lack of phenotype of MRP5−/− mice and the severe phenotype observed in worms and zebrafish is that a compensatory mechanism exists in mice. MRP9, the closest relative to MRP5, plays a compensatory role in heme homeostasis in the testes and mitochondrial metabolism.⁹⁵

BRCP/ABCG2, another member of the ABC transporter superfamily, has also been proposed as heme exporter. ABCG2 is one of the three major multidrug resistance proteins, which has led to its study in cancer. BCRP-1^−/− mice have protoporphyria, with a 10-fold increase of PP within erythrocytes. These mice become highly sensitive to the dietary chlorophyll-breakdown product pheophorbide, which is a structurally related to PP, resulting in extreme photosensitivity. A wide range of substrates has been shown for ABCG2, including drugs, xenobiotics, porphyrins, and heme.⁹⁶

Other ABC transporters also have been linked to heme homeostasis, such as ABCB-6, -7, -8, and -10, and are discussed in recent reviews.^80,96

2.3 |. Intracellular heme trafficking

After being synthesised in the mitochondria, heme must cross the inner and outer mitochondrial membranes to be incorporated into hemoproteins. As unbound heme is cytotoxic, there must be chaperones to bind and deliver heme to downstream target hemoproteins.⁹⁷ Intracellular heme can be divided into two pools, one that is relatively inert and stable, and a second that is labile. The first pool comprises high affinity heme binding molecules. Labile heme (LH) allows easy heme exchange among biomolecules with a modest heme affinity (Figure 3). The cytosol contains the largest LH pool, concentrations ranging from 20 to 340 nM, while the nucleus and mitochondria have less than 1 nM.^98,99 Although LH binding proteins remain largely unknown, several tools have been developed to measure LH based on fluorescence and activity-based sensors.^98–102

Several proteins have been shown to bind heme and play a role in heme homeostasis such as HBPs,¹⁰³ GAPDH,^104–106 FABP,¹⁰⁷ PGRMC, and GSTs.¹⁰⁸

GAPDH binds heme with Kd = 24 nM.¹⁰⁹ It regulates heme delivery and insertion into hemoproteins such as guanylate cyclase and NOS.^104–106 Each GAPDH tetramer binds one heme molecule.¹⁰⁹ GSTs catalyse the conjugation of GSH to electrophilic compounds,¹¹⁰ and also interact with porphyrins and heme.¹⁰⁸ The heme-binding proteins p22HBP, HBP1, and HBP23 bind both PP and heme. HBP23 loses its activity when it binds heme.¹⁰³ GSH binds free heme in a 1:1 ratio and converts heme to hematin (oxidised heme) in vitro,¹¹¹ preventing Fe2⁺-heme-induced oxidative damage. Thus, GSTs, FABPs, and HBPs all bind heme; however, their specific roles in heme homeostasis are still unclear.

PGRMC proteins 1 and 2, localize to the outer membrane of the mitochondria¹¹² and were recently shown to function as intracellular heme chaperones.^112–115 They interact with FECH the final enzyme of heme synthesis.¹¹² PGRMC2 is required for LH delivery to the nucleus, as the deletion of PGRMC2 in brown fat adipocytes decreased LH in the nucleus and increased stability of the heme-responsive transcriptional repressors Rev-Erbα and BACH1. Consequently, heme-regulated gene expression is altered, and this leads to severe mitochondrial defects and a failure to activate thermogenesis.³⁷

2.4 |. Circulating heme binding proteins

Intravascular hemolysis releases heme and haemoglobin into the bloodstream, such as in malaria, sickle cell disease (SCD), autoimmune hemolytic anaemia, and cancers.^116–123 In humans, serum albumin (HSA) and hemopexin (HPX) serve as heme scavengers.¹²⁴ The hemopexin-heme complex is endocytosed via CD91 receptors expressed in hepatocytes, macrophages, and neurons after which the hemopexin is recycled and the heme is degraded (Figure 3). Likewise, haemoglobin is bound by haptoglobin¹²⁵ and this complex is endocytosed by CD163 in macrophages and further degraded.⁷⁴

As mentioned previously, heme can also bind albumin which is the most abundant protein in the blood. The amounts of heme-albumin found regularly in human blood are in the range of 1 to 1.5 μM, and this level increases up to 50 μM during infection or inflammation. It has been proposed that TfR1 endocytoses the heme-albumin complex to deliver heme within cells.⁸⁴ While heme binds HSA, HPX has the highest affinity for heme with a Kd of 5 fM, whereas the HSA-heme Kd values are orders of magnitude higher, between 20 pM and 40 μM,^126–129 which makes HPX a preferential heme binding protein, albeit one of limited concentration and capacity.

Other proteins such as lipocalins, LDL/HDL, α1-microglobulin, and α1-antitrypsin are additional heme-binding proteins.⁹⁶ By measuring hemin dissociation rates from plasma proteins, after mixing a complex of hemin with each dansyl-labelled protein in vitro, Miller et al. concluded that 80% of hemin binds initially to LDL and HDL and then partially transfers to albumin and hemopexin.^124,130 The affinity of LDL and HDL to heme is lower than that of hemopexin with a Kd 10⁻¹¹ and 10⁻¹⁰ M respectively. The binding of hemin to LDL and HDL induces their oxidation and could contribute to atherosclerosis.

Diseases with severe hemolysis such as SCD release a large amount of free heme into the blood and have low circulating hemopexin levels.¹³¹ Excess of heme in the blood is cleared by kidneys and endothelial cells causing kidney diseases in hemolytic diseases.¹³² α-1-microglobulin, a small lipocalin protein with immunosuppressive functions found in plasma and tissues, scavenges heme, and targets it to the kidneys.¹³³

3 |. RELATION OF PORPHYRIN, IRON, AND HEME SYNTHESIS AND METABOLISM TO CELLULAR ENERGETICS METABOLISM

Heme and iron are essential cofactors for cellular energy production and oxygenation. The close relationship between heme synthesis and energy production is supported by multiple mitochondrial interactions with liver ALAS1 and bone marrow ALAS2.

3.1 |. Role of carbohydrates on ALAS1 in the liver: Lessons from hepatic porphyrias

A change in liver energy metabolism in AIP patients supported the induction of ALAS-1 worsening the symptoms of the disease and contributing to the persistence of clinical manifestations. Moreover, clinical expression of AIP is, at least in part, associated with a state of under-nutrition and the screening of serum hepatic proteins revealed that a significant number of AIP patients presented a decrease in insulin-like growth factor 1, transthyretin (prealbumin) or both.¹³⁴

In AHP the therapeutic effect of glucose loading [called the ‘glucose effect’] is well documented.¹³⁵ Historically, fasting is one of the well-known precipitating factors for AHP crisis,¹³⁶ and carbohydrate loading is widely used for both the treatment and prophylaxis. Glucose homeostasis during fasting is poorly understood in porphyrias. In an AIP mice model a different response to fasting was shown between carbohydrate metabolism in the liver and glucose consumption in the brain.¹³⁷ Moreover, the finding that AIP patients with acquired type 2 diabetes mellitus have fewer symptoms of AIP supports the protective role of elevated blood glucose levels.¹³⁸ The prominent role of insulin resistance in type 2 diabetes patients suggests the impact of reduced availability of energy metabolites in the severity of AIP pathophysiology. A dual therapy glucose plus insulin administration induces a synergic repressive effect on hepatic ALA synthase-1. In AIP mice, preventive treatment with an experimental fusion protein of insulin and apolipoprotein A1 improved the disease by promoting fat mobilisation in adipose tissue, increasing the metabolite bioavailability for the TCA cycle and inducing mitochondrial biogenesis in the liver.¹³⁹ By contrast, symptomatic AIP patients showed decreased insulin release and C-peptide levels in plasma associated with increased disease activity, suggesting that decreased glucose uptake by cells may accelerate heme synthesis.¹⁴⁰ Handschin et al. revealed that peroxisome proliferator-activated receptor gamma coactivator 1α [PGC1α], a transcriptional co-activator of nuclear receptors and other transcription factors, is a key player in porphyria induction during fasting and carbohydrate treatment (Figure 4).⁶⁴ They demonstrated that PGC1α increases ALAS1 mRNA levels. The combination of glucose and insulin was more potent in inhibiting fasting-mediated induction of PGC1α and ALAS1, suggesting that part of the beneficial effect of glucose in AHP attacks is mediated by the glucose-triggered increase of plasma insulin. During fasting, the induction of both PGC-1α and ALAS1 is due to glucagon action on the cAMP response element-binding transcriptional factor, which binds directly to the PGC1α and ALAS1 promoters.¹⁴¹ Conversely, the insulin pathway involving protein kinase B (Akt) in the liver inhibits ALAS1 transcription.⁶⁵ This ALAS1 down-regulation is a consequence of the phosphorylation of the transcriptional factor Forkhead box protein O1 (FOXO1) induced by Akt activation, which disrupts binding to PGC1α.¹⁴² Altogether, the activation of ALAS1 expression by PGC1α is a consequence of the co-activation of FOXO1, which binds to the insulin-response element in the promoter of ALAS1 (Figure 4).¹⁴³ Elevated blood lactate concentration, a classical hallmark of mitochondrial electron transport chain (ETC) dysfunction, was found in AIP patients compared to controls, suggesting that AIP patients may be affected by a glycolytic-ETC defect.¹⁴⁴

FIGURE 4.

Effect of fasting and glucose intake on ALAS1 transcription. The PGC-1a and FOXO-1 bind together with the insulin responsive element site on the ALAS-1. During fasting, the induction of PGC-1α is due to glucagon action through its receptor with consequent ATP reduction by adenylate cyclase in cAMP in the cytosol of hepatocytes. Conversely, the insulin pathway involving protein kinase B (Akt) in the liver inhibits ALAS1 transcription because of the transcriptional factor Forkhead box protein O1 (FOXO1) phosphorylation which disrupts binding to PGC-1α. (adapted and amended from Di Pierro and Granata. Nutrients and Porphyria: An Intriguing Crosstalk. Int J Mol Sci. 2020;21(10):3462. doi: 10.3390/ijms21103462.).

3.2 |. Crosstalk between ALAS1 and the TCA cycle and ETC

Both heme and iron are essential as cofactors in the structure of proteins involved in oxidative phosphorylation and anti-oxidative enzymes.^136,145 Briefly, heme ring is a constitutive element of cytochromes involved in terminal complexes (cytochrome c and complex IV) of the ETC, whereas iron–sulfur clusters are involved in initial complexes I, II and III (Figure 5). ALAS depends on the TCA cycle for its substrate, succinyl-CoA, which is exclusively formed within the mitochondria by the enzyme succinyl-CoA synthetase (SCoAS). There are two SCoAS isoforms: a GTP- and an ATP-specific isoform. Their β-subunits differ, and this determines the specificity of nucleotide binding.¹⁴⁶ The next enzymatic step in the TCA cycle, rewires succinate dehydrogenase (SDH) or ETC complex II activity, and is shared by both the ETC and the TCA cycle (Figure 5). The ALAS1-TCA-ETC relationship in hepatocytes has been studied in a HMBS^−/− deficient mouse model and reveals a failure of mitochondrial energetic metabolism in the liver during the AIP crises induced by phenobarbital; the resolution of the crises was accompanied by partial or complete recovery.^147,148 Surprisingly, no defect affecting the three terminal ETC components containing heme as prosthetic group was observed, even though the activities of iron–sulfur cluster complexes were affected. Indeed, complex II-driven respiration, which is activated by succinyl-CoA, is directly impacted by the available supplies from the TCA intermediates and was found to be functionally affected. The TCA cycle was greatly affected at two levels, either the synthesis of succinyl-CoA or its utilisation via α-ketoglutarate dehydrogenase (α-KGDH) and SDH. KGDH activity depends on α-lipoic acid which biosynthesis requires a Fe-S cluster. Interestingly, in human sideroblastic anaemia, Daher et al. demonstrated that mutations in the glutaredoxin-5 (GLRX5) gene, which encoded a protein involved in Fe-S cluster assembly, drastically decrease the lipoic acid-ratio both in KGDH and in pyruvate dehydrogenase (PDH). As a consequence of this low level of lipoic acid, succinyl CoA availability was reduced leading to a significant drop in ALAS2 activity and a very low erythroid heme synthesis.¹⁴⁹ Moreover, in a hepatocyte cell line transfected with interfering RNA targeting HMBS, the insulin-mimicking α-lipoic acid improved glucose metabolism and mitochondrial dysfunction. Lipoic acid thus improved heme biosynthesis, glucose metabolism, and hyperinsulinemia occurring in fasted AIP mice.¹⁵⁰ The sharp increase of ALAS1 activity during the AIP crises may deplete most of the succinyl-CoA available in the mitochondria. Consequently, the acute depletion of this intermediate metabolite could alter the metabolite flux through the TCA cycle, a phenomenon known as cataplerosis, which is usually compensated by anaplerotic reactions that counter the lack of the metabolite. Cataplerosis leads to the incapacity of the TCA cycle to generate enough reduced substrates to fuel the ETC [Figure 4]. The administration of carbohydrates increases the availability of acetyl-CoA for the TCA cycle by up-regulating citrate synthase activity and thereby the metabolic flux across the TCA cycle. Heme and siRNA targeting ALAS1 both down-regulate ALAS1 and subsequently increase the availability of succinyl-CoA for the TCA cycle and the NADH/H⁺, FADH₂ supply for the ETC. Recently, a preliminary report underlined the relationship between mitochondrial cell number and heme biosynthesis. The mitochondrial DNA copy number and serum levels of PERM1, a marker of mitochondrial biogenesis, were found lower in AIP patients compared to healthy controls suggesting a low number of mitochondria. This could suggest some adaptative and protective mechanisms against an inherited heme synthesis deficiency.¹⁵¹

FIGURE 5.

Cata/Anaplerosis—Interconnections among Heme Synthesis, and Energetic Metabolism—the Mitochondrial Electron Transport Chain [ETC], the Tricarboxylic Acid Cycle [TCA], Glycolysis, and Beta Oxidation. Involvement of heme and iron as co-factors in proteins of oxidative phosphorylation and TCA cycle. The transport of acyl-CoA into mitochondria, facilitated by the two carnitine palmitoyl transferases, CPT1 and CPT2, is followed by β-oxidation, providing acetyl-CoA to the TCA cycle. Acetyl-CoA is also supplied to the TCA cycle by pyruvate dehydrogenase (PDH), which oxidises the pyruvate produced by cytoplasmic glycolysis. The TCA cycle provides NADH/H+ and FADH2 for the mitochondrial ETC and oxidative phosphorylation. ALAS1 depends on the TCA cycle for its substrate, succinyl-CoA. (A) At the basal level with uninduced ALAS1, normal heme synthesis allows for normal hepatic cellular energy production by the mitcohondrial electron transport chain and linked ATP production. Treatment of AIP attacks with heme arginate or ALAS1siRNA reduces the withdrawal of succinyl-CoA, while treatment with glucose increases its supply. (B) ALAS1 overexpression (as occurs during AIP attacks) is envisioned to induce the withdrawal of succinyl-CoA from the TCA cycle into synthesis of ALA, impairing the TCA and ETC fluxes.

The control of cell energy metabolism and of nutrient expenditure to sustain energy production is particularly important under conditions of high-energy demand, such as proliferation. Indeed, FLVCR1a is overexpressed in several tumour types. Recently, a heme synthesis - FLVCR1a export axis was identified as a key regulator of the TCA cycle and oxidative metabolism.¹⁵² Conversely, inhibition of heme synthesis promotes TCA cycle flux and oxidative phosphorylation. Therefore, alterations in heme metabolism are frequently observed in cancer.¹⁵³ It is commonly assumed that most tumours rely on high heme synthesis by ALAS1. In the near future, several components or indirect modulators of the TCA cycle may be explored for therapeutic purposes. Although high toxicity remains an issue, some of those approaches, including ALAS1 siRNA have been shown to be well tolerated clinically.^154,155 Some drugs expected to inhibit/enhance heme synthesis and heme export are in development and could be used to interfere with cell metabolic adaptation in various disease types encompassing cancer, obesity, neurodegenerative, disorders, infections, muscular diseases, and diabetes.

Key points.

• Heme (iron protoporphyrin) is a primordial macrocycle upon which nearly all aerobic life on Earth depends.

• The first and normally rate-controlling step of heme synthesis is catalysed by 5-aminolevulinate synthase [ALAS].

• Abnormalities in enzymes of heme synthesis give rise to porphyrias.

• Recent evidence establishes the presence of heme transporters suggesting that cells and organs may share heme.

• Heme and iron, although promoters of oxidative stress and potentially toxic, are essential cofactors for cellular energy production and oxygenation. The close relationship between heme synthesis and energy production is supported by multiple mitochondrial interactions with liver ALAS1 and bone marrow ALAS2.

Abbreviations:

ABC

ATP-binding cassette

ADP

ALA dehydratase deficiency porphyria

AHP

acute hepatic porphyria[s]

AIP

acute intermittent porphyria

ALA

5-aminolevulinic acid

ALAD

ALA dehydratase

ATP

adenosine tri-phosphate

BACH

BTB and CNC homology transcription factor/suppressor

BRCP

breast cancer resistance protein

BTB

broad-complex tram track, bric-a-brac

CAR

constitutive androstane receptor

CD

cluster designation

ClpX

an ATP-dependent protein chaperone and peptidase that helps maintain protein quality

CNC

cap-’n-collar

CO

carbon monoxide

CP

cysteine-proline

CPOX

coproporphyrinogen oxidase

CPT

carnitine-palmitoyl transferase

CRISPR

clustered regularly interspaced short palindromic repeats

CYP

cytochrome P-450

DGCR8

DiGeorge syndrome critical region 8

DMT1

divalent metal transporter-1

EGR

epidermal growth factor

ELK

E-26 oncogene

EPP

erythropoietic protoporphyria

ER

endoplasmic reticulum

E[S]RR

oestrogen-related receptor-alpha

ETC

electron transport chain of mitochondria

FABP

fatty acid-binding protein

FECH

ferrochelatase

FLVCR

feline leukaemia virus group C receptor

FOX01

forkhead box protein-01, a transcription factor

FPN

ferroportin

GAPDH

glyceraldehyde phosphate dehydrogenase

GATA

GATA-binding factor, aka erythroid transcription factor

GLRX5

glutaredoxin-5

GSH

glutathione-reduced

GST[s]

glutathione-S-transferase[s]

HBPs

heme binding proteins

H

heavy chain of ferritin

HCP

hereditary coproporphyria

HCP1

heme carrier protein-1

HDL

high-density lipoprotein

HeLa

immortal and oldest human cancer cell line derived from cervical cancer of Henrietta Lacks

HEK

human embryo kidney

HEP

hepato-erythropoietic porphyria

HIF

hypoxia inducible factor

HMB[S]

hydroxymethylbilane [synthase]

HMOX

heme oxygenase

HPX

hemopexin

HRG

heme responsive gene[s]

HSA

human serum albumin

IRE

iron regulatory element[s]

IRP

iron regulatory protein[s]

KGDH

alpha-keto glutarate dehydrogenase

L

light chain of ferritin

LDL

low-density lipoprotein

LH

Labile heme

LRP

low density lipoprotein-related protein

MAMs

mitochondria-associated membranes

MCSs

membrane contact sites

MDR

multi-drug resistance

MePNs

metalloporphyrins

MFSD7c

aka FLVCR2, an orphan transporter

MRP

multidrug resistant protein

NF-E2

nuclear factor-E2

NOS

nitric oxide synthase

NR1D1

nuclear receptor subfamily 1 group D member 1

OATP

organic anion transport protein

PBG

porphobilinogen

PBGD

PBG deaminase

PC

phosphatidylcholine

PCF

post coupled folate

PCT

porphyria cutanea tarda

PDH

pyruvate dehydrogenase

PERM 1

PGC-1/E[S] RR-induced regulator in muscle 1

PGRMC[s]

progesterone membrane receptor component[s]

PGC1α

peroxisome proliferator-activated receptor gamma coactivator 1α

PLP

pyridoxal phosphate

PP

protoporphyrin IX

PPAR

peroxisome proliferator associated receptor

PPOX

protoporphyrinogen 9 oxidase

PXR

pregnane X receptor

RES

reticuloendothelial system

Rev-Erb

a heme-binding nuclear receptor, product of the NR1D1 gene that is involved in circadian rhythms

SCD

sickle cell disease

SLCO2B1

solute carrier organic anion transporter family member 2B1, aka OATP2B1

SA

sideroblastic anemias

SCoAS

succinyl-CoA synthetase

SDH

succinate dehydrogenase

TCA

tricarboxylic acid

TfR1

transferrin receptor-1

TMD

transmembrane domain

UROD

uroporphyrinogen decarboxylase

URO3S

uroporphyrinogen 3 synthase

UTR

untranslated region[s]

VP

variegate porphyria

XLP

X-linked protoporphyria

ZnPP

zinc protoporphyrin