Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.     

X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.     

Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model

The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1(+ex2)) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1(+ex2) homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.     

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.     

Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans

5-Aminolevulinate synthase (ALAS) is the first and rate-limiting enzyme of heme biosynthesis in humans, animals, other non-plant eukaryotes, and alpha-proteobacteria. It catalyzes the synthesis of 5-aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl-coenzyme A (sCoA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. X-linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALAS(Rc)) binding its cofactor PLP. We, furthermore, present structures of ALAS(Rc) in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA-causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.     

Multiple regulatory steps in erythroid heme biosynthesis

Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent down-stream rate-limiting steps.     

Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition.

5-Aminolevulinate synthase (ALAS; E.C. 2.3.1.37) catalyzes the first and rate-limiting step of heme synthesis within the mitochondria. Two isozymes of ALAS, encoded by separate genes, exist. ALAS1 is ubiquitously expressed and provides heme for cytochromes and other hemoproteins. ALAS2 is expressed exclusively in erythroid cells and synthesizes heme specifically for haemoglobin. A database search for proteins potentially regulated by oxygen tension revealed that ALAS2 contained a sequence of amino acids (LXXLAP where L is leucine, X is any amino acid, A is alanine, and P is proline) not occurring in ALAS1, which may be hydroxylated under normoxic conditions (21% O2) and target the enzyme for ubiquitination and degradation by the proteasome. We examined protein turnover of ALAS2 in the presence of cycloheximide in K562 cells. Normoxic ALAS2 had a turnover time of approximately 36 h. Hypoxia (1% O2) and inhibition of the proteasome increased both the stability and the specific activity of ALAS2 (greater than 2- and 7-fold, respectively, over 72 h of treatment). Mutation of a key proline within the LXXLAP sequence of ALAS2 also stabilized the protein beyond 36 h under normoxic conditions. The von Hippel-Lindau (vHL) protein was immunoprecipitated with FLAG epitope-tagged ALAS2 produced in normoxic cells but not in hypoxic cells, suggesting that the ALAS2 is hydroxylated under normoxic conditions and targeted for ubiquitination by the E3 ubiquitin ligase system. ALAS2 could also be ubiquitinated under normoxia using an in vitro ubiquitination assay. The present study provides evidence that ALAS2 is broken down under normoxic conditions by the proteasome and that the prolyl-4-hydroxylase/vHL E3 ubiquitin ligase pathway may be involved.     

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.     

Identification of a splice variant of mouse caveolin-2 mRNA encoding an isoform lacking the C-terminal domain

We identified a splice variant of mouse caveolin-2 mRNA having an intronic sequence in place of the third exon (Deltaex3). The entire sequence of full-length (FL) and Deltaex3 caveolin-2 mRNA was determined; their sizes were 2490 and 973 bp, respectively. The Deltaex3 mRNA encoded a putative isoform lacking the C-terminal 49 amino acids of the authentic caveolin-2. The expression level of Deltaex3 was lower than that of FL mRNA, but considerable in some culture cells and tissues. The isoform lacking the C-terminus localized to the endoplasmic reticulum, while the authentic caveolin-2 was distributed to the Golgi and the plasma membrane along with caveolin-1. The result confirmed the necessity of the C-terminal domain of caveolin-2 for the caveolar localization, and showed the existence of a novel caveolin-2 isoform, which is not recruited to caveolae even in the presence of caveolin-1.     

Structure of a mouse erythroid 5-aminolevulinate synthase gene and mapping of erythroid-specific DNAse I hypersensitive sites.

The enzyme 5-aminolevulinate synthase (ALA-S) catalyzes the first step in heme biosynthesis. In this study, the mouse erythroid gene has been cloned and analyzed in order to investigate the regulation of ALA-S expression during erythroid differentiation. The gene spans approximately kbp and consists of 11 exons and 10 introns. The first exon is 37 bp, non-coding, and followed by a 6kb intron. The mRNA capsite was mapped by primer extension and defines a promoter that contains no apparent TATA element. S1 nuclease analysis detects the presence at low levels of a 45 bp-deleted form of the ALA-S mRNA created by the use of an alternative splice site at the intron 2/exon 3 junction. Five DNAse I hypersensitive sites were detected in chromatin from uninduced and induced MEL cells. One site is at the promoter; the others are in the body of the gene. No significant differences were observed in the patterns or intensity of the hypersensitive sites in the uninduced and induced MEL cells, however, no sites in ALA-S were observed in NIH 3T3 cells or in deproteinized DNA. Thus, these sites are specific for erythroid chromatin but appear to be established at an earlier stage of differentiation than represented by the uninduced MEL cell.