The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase

The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and (1)H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H(2)O solutions and micellar environments. A well defined amphipathic alpha-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short alpha-helix, defined by A5-L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and (1)H NMR study of hemin and psALAS indicated that a heme-peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.     

Structural environment dictates the biological significance of heme-responsive motifs and the role of Hsp90 in the activation of the heme activator protein Hap1

Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins. The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1. To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1. We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90. Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90. These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes. The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence. Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction. It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation. Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction.     

A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.

Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.     

Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian Cell Death

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.     

Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys¹¹⁴ as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys–Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys–Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H₂O₂ induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.     

Regulation of heme synthesis in the regenerating rat liver

This investigation shows that the regulation of heme synthesis in the regenerating rat liver does not differ from the regulation in the normal liver. The heme saturation of tryptophan pyrrolase was found to be low, indicating a reduced concentration of heme in the regulatory heme pool of the regenerating rat liver. As expected, ALAS in the mitochondrial fraction was found to be elevated. It was also shown that ALAS in the regenerating rat liver can be induced by the porphyrinogenic drugs AIA and DDC and that heme reduces its activity. The decrease observed in the activity of cytosolic ALAS might be due to impaired synthesis of the enzyme but does not affect the regulation of the heme biosynthetic pathway.     

Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase.

During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.     

Free heme pool and activity of key enzyme of heme synthesis in the rat liver under action of agents affecting reduced glutathione level

The decrease of GSH level in the rat liver was found to be accompanied by an increase of tryptophan 2,3-dioxygenase (TDO) heme saturation during first hours after HgCl2, phenylhydrazine (Ph) injection or rhabdomyolysis (the coefficient of correlation -0.978). The activity of the key enzyme of heme synthesis--5-aminolevulinate synthase (ALAS) was 2.5-fold increased in the first hours after Ph injection and rhabdomyolysis. Glutathione injection in vivo as well as CdCl2 caused the increase of GSH content and the inhibition of ALAS. The coefficient of correlation for GSH content and ALAS activity under the action of agents altering both these parameters (CdCl2, Ph, GSH injection and rhabdomyolysis) is 0.938. Taking into account the presence of heme regulatory motif with conserved cystein in many proteins, including ALAS and TDO (accession number in SwissProt database AAH61793 and P21643, respectively), the link between alterations of GSH content, ALAS activity and heme saturation of TDO in the rat liver could be proposed. The further experiments should be performed in order to elucidate the mechanisms of GSH level influence on free heme pool formation in the liver cells.     

The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx^−/− cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism.     

Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin

5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60-70%, but those of the cytosolic precursor protein were up-regulated by 2-5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.