Brian K. May

Molecular Regulation of Heme Biosynthesis in Higher Vertebrates

Publisher Summary The regulation of heme biosynthesis in animals has been a topic of interest for many years. It is generally agreed that the first enzyme of the heme pathway, 5-aminolevulinate synthase, determines the rate of heme biosynthesis and the regulation of this enzyme is, therefore, a major focus of this chapter. The heme biosynthetic pathway is present in all cell types except mature erythrocytes. The final step of heme biosynthesis occurs in mitochondria and the heme is then utilized for the formation of different hemoproteins located in mitochondria, microsomes, peroxisomes, the cytosol, and probably the nucleus. Heme can control the biosynthesis of some proteins. In erythroid cells, heme controls the translation of proteins, notably α- and β-globin chains, by modulating the activity of a specific kinase. All nucleated animal cells must synthesize heme for incorporation into respiratory cytochromes, but erythroid and liver cells have the highest rates of heme synthesis. Erythroid cells synthesize about 90% of the total heme in the body for assembly into hemoglobin. Although the bulk of heme in the liver is made in situ , the liver may also obtain some heme from serum haptoglobin–hemoglobin and heme–hemopexin complexes, following intravascular hemolysis. All enzymes of the heme biosynthetic pathway, except for protoporphyrinogen oxidase, have been cloned from higher vertebrates. The genes encoding these enzymes are located on different chromosomes.

Transcriptional Synergism between Vitamin D-responsive Elements in the Rat 25-Hydroxyvitamin D3 24-Hydroxylase (CYP24) Promoter

Transcription of the CYP24 gene is induced by 1,25-(OH)2D3 through a vitamin D receptor-dependent process. The functional activities of three possible vitamin D response elements (VDREs), located on the antisense strand of the rat CYP24 promoter, were investigated by transient expression of native and mutant promoter constructs in COS-1, JTC-12, and ROS 17/2.8 cells. A putative VDRE with a half-site spacing of 6 base pairs at −249/−232 (VDRE-3) did not contribute to 1,25-(OH)2D3 induced expression in the native promoter, although activity has been reported when the element was fused to the heterologous thymidine kinase promoter. Two VDREs with half-site spacings of 3 base pairs at −150/−136 and −258/−244 (VDRE-1 and VDRE-2, respectively), showed transcriptional synergism in COS-1 cells when treated with 1,25-(OH)2D3 (10−7 to 10−11 M). The contribution of both VDREs was hormone-concentration dependent from 10−10 to 10−12 M, with VDRE-1 demonstrating greatest sensitivity to 1,25-(OH)2D3. Transactivation by VDRE-1 was always greater than VDRE-2, but the converse was observed for the binding of vitamin D receptor-retinoid X receptor complex by each VDRE in gel mobility shift assays. The synergy observed between VDRE-1 and VDRE-2 may have important implications in cellular responses to different circulating levels of 1,25-(OH)2D3.

X-linked pyridoxine-responsive sideroblastic anemia due to a Thr388-to-Ser substitution in erythroid 5-aminolevulinate synthase

BACKGROUND X-linked sideroblastic anemia is usually associated with reduced 5-aminolevulinate synthase activity in erythroid cells, and some cases are responsive to treatment with pyridoxine, the precursor to the cofactor of the enzyme. The recently identified gene for an erythroid-specific 5-aminolevulinate synthase isoenzyme and its localization to the X chromosome make it likely that one or more defects in this gene underlie the anemia. METHODS Using a polymorphic dinucleotide-repeat sequence in the erythroid 5-aminolevulinate synthase gene, we confirmed the linkage of this gene to the disorder in a family with X-linked pyridoxine-responsive sideroblastic anemia. We therefore sought evidence of a nucleotide-sequence abnormality in the erythroid 5-aminolevulinate synthase gene by analyzing enzymatically amplified DNA. RESULTS DNA-sequencing studies in two affected males and one carrier female in the kindred demonstrated a cytosine-to-guanine change at nucleotide 1215 (in exon 8). This change results in the substitution of serine for threonine at amino acid residue 388, near the lysine that binds the pyridoxal phosphate cofactor. In expression studies, the activity of the mutant enzyme was reduced relative to that of the wild type, and this reduction was comparable to that in erythroid cells of the proband during relapse of the anemia; the enzyme activity expressed in the presence of pyridoxine was comparable to that in the probands marrow cells during remission. Although the affinity of the mutant enzyme for pyridoxal phosphate was not altered, the mutation appears to introduce a conformational change at the active site of the enzyme. CONCLUSIONS We identified a point mutation resulting in an amino acid change near the pyridoxal phosphate-binding site of the erythroid 5-aminolevulinate synthase isoenzyme as the underlying defect in a kindred with X-linked pyridoxine-responsive sideroblastic anemia.

Overview of regulatory cytochrome P450 enzymes of the vitamin D pathway

Cytochromes P450c1 and P450c24 are regulated hydroxylase enzymes that direct the bioactivation and metabolic degradation of vitamin D. The bioactivation pathway is regulated by cytochrome P450c1 through its synthesis of 1alpha,25(OH)(2)D(3), the hormonally active form of the vitamin. Expression of the P450c1 gene is regulated at the transcription level. Promoter regions within the P450c1 gene have been identified that respond to cAMP and 1alpha,25(OH)(2)D(3) during the respective up- and down-regulation of P450c1 gene expression. The diametric action of 1alpha,25(OH)(2)D(3) to up-regulate P450c24 gene expression is discussed in the context of two vitamin D response elements (VDREs) that are linked functionally to an adjoining Ets-binding site. It is apparent from sequence-derived data that the P450c1 and P450c24 enzymes share only 10-25% sequence identity, yet they display functionally similar domains that are conserved across the family of cytochrome P450 enzymes. Expression of E. coli recombinant P450c1 and P450c24 enzymes, and the substrate-binding parameters for P450c24 are discussed. Finally, the natural point mutations in human P540c1 from patients with pseudovitamin D-deficiency rickets (PDDR) are discussed in the context of the enzymes structure and function.

Transcriptional activation of cytochrome P450 genes by different classes of chemical inducers.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes.

Vitamin D Depletion Induces RANKL-Mediated Osteoclastogenesis and Bone Loss in a Rodent Model†

The association between increased risk of hip fracture and low vitamin D status has long been recognized. However, the level of vitamin D required to maintain bone strength is controversial. We used a rodent model of vitamin D depletion to quantify the 25‐hydroxyvitamin D (25D) levels required for normal mineralization. Six groups of 10‐wk‐old male Sprague‐Dawley rats (n = 42) were fed a diet containing 0.4% calcium and various levels of dietary vitamin D3 for 4 mo to achieve stable mean serum 25D levels ranging between 10 and 115 nM. At 7 mo of age, animals were killed, and the histomorphometry of distal and proximal femora and L2 vertebra was analyzed. Total RNA was extracted from whole femora for real‐time RT‐PCR analyses. In the distal femoral metaphysis, trabecular bone mineral volume (BV/TV) showed a significant positive association with circulating 25D levels (r2 = 0.42, p < 0.01) in the animals with serum 25D levels between 20 and 115 nM. Osteoclast surface (Oc.S) levels were positively associated with RANKL:OPG mRNA ratio, higher in groups with lower serum 25D levels, and were independent of serum 1,25D levels. Serum 25D levels <80 nM gave rise to osteopenia as a result of increased osteoclastogenesis, suggesting that levels of 25D >80 nM are needed for optimal bone volume. These data indicate that serum 25D levels are a major determinant of osteoclastogenesis and bone mineral volume and are consistent with the levels of 25D recommended to reduce the risk of fracture in humans.

Regulation of erythroid 5-aminolevulinate synthase expression during erythropoiesis.

Erythroid tissue is the major site of heme production in the body. The synthesis of heme and globin chains is coordinated at both the transcriptional and post-transcriptional levels to ensure that virtually no free heme or globin protein accumulates. The key rate-controlling enzyme of the heme biosynthetic pathway is 5-aminolevulinate synthase (ALAS) and an erythroid-specific isoform (ALAS2) is up-regulated during erythropoiesis. Differentiation of embryonic stem cells with a disrupted ALAS2 gene has established that expression of this gene is critical for erythropoiesis and cannot be compensated by expression of the ubiquitous isoform of the enzyme (ALAS1). Interestingly, heme appears to be important for expression of globin and other late erythroid genes and for erythroid cell differentiation although the mechanism of this effect is not clear. Transcriptional control elements that regulate the human gene for ALAS2 have been identified both in the promoter and in intronic enhancer regions. Subsequent translation of the ALAS2 mRNA is dependent on an adequate iron supply. The mechanism by which transcription of the gene for ALAS2 is increased by erythropoietin late in erythropoiesis remains an interesting issue. Erythropoietin action may result in altered levels of critical erythroid transcription factors or modulate the phosphorylation/acetylation status of these factors. Defects in the coding region of the gene for ALAS2 underlie the disease state X-linked sideroblastic anemia. In this review, we focus on the regulation and function of erythroid-specific 5-aminolevulinate synthase during erythropoiesis and its role in the X-linked sideroblastic anemia.

Characteristics of extracellular protease formation by Bacillus subtilis and its control by amino acid repression

Abstract 1. The formation of extracellular protease has been studied in washed-cell suspensions of Bacillus subtilis . Formation of the enzyme is inhibited by chloramphenicol and actinomycin D; its production is therefore due to de novo synthesis of the enzyme. 2. An examination of the distribution of protease between extra- and intracellular phases in washed cells showed the absence of any significant pool of preformed enzyme accumulating inside the cell over a 3-h period. 3. The rate of protease synthesis (but not of the simultaneously produced exoenzyme, α-amylase) is strongly repressed by the amino acids in the medium. No single amino acid is completely effective, but a mixture of either isoleucine and proline or of glutamic acid, aspartic acid, glutamine and asparagine gives maximal repression of protease formation.

Control of 5-aminolevulinate synthase in animals

The proposed mechanism by which hepatic ALV-synthase mitochondrial levels are regulated is outlined in Fig. 2. ALV-synthase catalyzes the first and rate-limiting step in the heme pathway and is normally present in low amounts. A cytosolic, regulatory free heme pool tightly controls the amount of ALV-synthase in two ways. In the primary mechanism of regulation, heme is proposed to inhibit the synthesis of ALV-synthase mRNA. Most likely this would be mediated through the action of specific heme-binding protein(s) which recognize regulatory control regions of the ALV-synthase gene. Gene activity therefore is significantly repressed most of the time. When there is an increased demand for heme by newly synthesized cellular hemoproteins, the free heme pool is reduced, leading to a derepression of ALV-synthase mRNA synthesis. Once the need for increased heme synthesis is satisfied, inhibitory heme levels build up again. When drugs such as phenobarbital are administered to animals, there is a rapid induction in the liver of both cytochrome P-450 and ALV-synthase. It is proposed that the heme pool governing ALV-synthase levels is lowered by the increased heme demand due to cytochrome P-450 apoprotein formation. The primary event in the drug induction of ALV-synthase is therefore the increased synthesis of cytochrome P-450 apoprotein. However, the mechanism by which this occurs is unknown, although drugs do increase the synthesis of mRNA for cytochrome P-450 (Fig. 2). (There is evidence that for the aromatic hydrocarbons a specific cytosolic receptor exists.) In the acute hepatic porphyria diseases, uncontrolled synthesis of hepatic ALV-synthase occurs. The various forms are characterized by reduced levels of one of the heme pathway enzymes other than ALV-synthase. Attacks of the disease are commonly precipitated by drugs which induce cytochrome P-450, and the uncontrolled accumulation of ALV-synthase which accompanies these attacks results from the combined action of the block in the heme pathway and the increased cytochrome P-450 levels. A major challenge which now exists is to understand at the molecular level how the genes for ALV-synthase and cytochrome P-450 are regulated in the liver and other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Transcriptional Regulation of the Human Erythroid 5-Aminolevulinate Synthase Gene IDENTIFICATION OF PROMOTER ELEMENTS AND ROLE OF REGULATORY PROTEINS

We have characterized the 5′-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at −126/−121 (on the noncoding strand) and −102/−97 were each recognized by GATA-1 proteinin vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (−30/−23) bound GATA-1- or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the −30/−23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at −59/−48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Krüppel-like factor, and basic Krüppel-like factor in vitro, while in transactivation experiments erythroid Krüppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at −49/−39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5′-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo.