Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 µL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

Murine models of erythroid 5ALA synthesis disorders and their conditional synthetic lethal dependency on pyridoxine.

ABSTRACT: X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase (ALAS), ALAS2, which encodes the first enzyme in heme biosynthesis. A related congenital sideroblastic anemia (CSA) is due to mutations in SLC25A38 (solute carrier family 25 member A38), which supplies mitochondrial glycine for ALAS2 (SLC25A38-CSA). The lack of viable animal models has limited the studies on pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knockin mouse models that recapitulate the main features of XLSA and XLPP; and using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP/EPP protoporphyrias, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ ß-thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis-related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases.

Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease.

The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.

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

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.

From chemistry to genomics: A concise history of the porphyrias.

We describe developments in understanding of the porphyrias associated with each step in the haem biosynthesis pathway and the role of individuals whose contributions led to major advances over the past 150 years. The first case of erythropoietic porphyria was reported in 1870, and the first with acute porphyria in 1889. Photosensitisation by porphyrin was confirmed by Meyer-Betz, who self-injected haematoporphyrin. Günther classified porphyrias into haematoporphyria acuta, acuta toxica, congenita and chronica. This was revised by Waldenström into porphyria congenita, acuta and cutanea tarda, with the latter describing those with late-onset skin lesions. Waldenström was the first to recognise porphobilinogen's association with acute porphyria, although its structure was not solved until 1953. Hans Fischer was awarded the Nobel prize in 1930 for solving the structure of porphyrins and the synthesis of haemin. After 1945, research by several groups elucidated the pathway of haem biosynthesis and its negative feedback regulation by haem. By 1961, following the work of Watson, Schmid, Rimington, Goldberg, Dean, Magnus and others, aided by the availability of modern techniques of porphyrin separation, six of the porphyrias were identified and classified as erythropoietic or hepatic. The seventh, 5-aminolaevulinate dehydratase deficiency porphyria, was described by Doss in 1979. The discovery of increased hepatic 5-aminolaevulinate synthase activity in acute porphyria led to development of haematin as a treatment for acute attacks. By 2000, all the haem biosynthesis genes were cloned, sequenced and assigned to chromosomes and disease-specific mutations identified in all inherited porphyrias. These advances have allowed definitive family studies and development of new treatments.

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Comprehensive Genomic Analysis Identifies a Diverse Landscape of Sideroblastic and Nonsideroblastic Iron-Related Anemias with Novel and Pathogenic Variants in an Iron-Deficient Endemic Setting.

Inherited iron metabolism defects are possibly missed or underdiagnosed in iron-deficient endemic settings because of a lack of awareness or a methodical screening approach. Hence, we systematically evaluated anemia cases (2019 to 2021) based on clinical phenotype, normal screening tests (high-performance liquid chromatography, α gene sequencing, erythrocyte sedimentation rate, C-reactive protein, and tissue transglutaminase), and abnormal iron profile by targeted next-generation sequencing (26-gene panel) supplemented with whole-exome sequencing, multiplex ligation probe amplification/mitochondrial DNA sequencing, and chromosomal microarray. Novel variants in ALAS2, STEAP3, and HSPA9 genes were functionally validated. A total of 290 anemia cases were screened, and 41 (14%) enrolled for genomic testing as per inclusion criteria. Comprehensive genomic testing revealed pathogenic variants in 23 of 41 cases (56%). Congenital sideroblastic anemia was the most common diagnosis (14/23; 61%), with pathogenic variations in ALAS2 (n = 6), SLC25A38 (n = 3), HSPA9 (n = 2) and HSCB, SLC19A2, and mitochondrial DNA deletion (n = 1 each). Nonsideroblastic iron defects included STEAP3-related microcytic anemia (2/23; 8.7%) and hypotransferrenemia (1/23; 4.3%). A total of 6 of 22 cases (27%) revealed a non-iron metabolism gene defect on whole-exome sequencing. Eleven novel variants (including variants of uncertain significance) were noted in 13 cases. Genotype-phenotype correlation revealed a significant association of frameshift/nonsense/splice variants with lower presentation age (0.8 months versus 9 years; P < 0.01) compared with missense variants. The systematic evaluation helped uncover an inherited iron defect in 41% (17/41) of cases, suggesting the need for active screening and awareness for these rare diseases in an iron-deficient endemic population.

Gene expression analysis revealed downregulation of complement receptor 1 in clonal B cells in cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.

Regulatory role of ceRNA network in B lymphocytes of patients with immune thrombocytopenia.

OBJECTIVE: High-throughput sequencing was used to screen expressing differences of miRNA, lncRNA, and mRNA in CD19+ B peripheral blood samples of newly diagnosed immune thrombocytopenia (ITP) patients and healthy controls. The study aimed to explore the regulatory role of ceRNA network in the pathogenesis of dysfunctional CD19 + B lymphocytes of ITP patients. METHODS: CD19+ B lymphocytes were isolated from peripheral blood samples of ITP patients and their healthy counterparts. High-throughput sequencing was used to screen for the expression of miRNA, lncRNA, and mRNA of ITP patients and healthy controls, which were analysed by the ceRNA network. Moreover, qPCR was used to verify the differential expression of miRNA, lncRNA, and mRNA in ITP patients and healthy controls. The correlation between differentially expressed miRNA, lncRNA, mRNA, and B lymphocyte subsets was also analysed. RESULTS: The CD19+ B lymphocytes of 4 newly diagnosed ITP patients and 4 healthy controls were sequenced and analysed. There were 65 differentially expressed lncRNA and 149 mRNA forming a ceRNA network showed that 12 lncRNA and 136 differentially expressed mRNA were closely associated. Similarly, miR-144-3p, miR-374c-3p, and miR-451a were highly expressed in ITP patients, as confirmed by qPCR, which was consistent with the high-throughput sequence results. LOC102724852 and CCL20 were highly expressed in ITP patients, while LOC105378901, LOC112268311, ALAS2, and TBC1D3F were not as compared to healthy controls, which was consistent with the high-throughput sequence results. In addition, the expression of miR-374c-3p, LOC112268311, LOC105378901, and CXCL3 were correlated with the percentage of B lymphocyte subsets. CONCLUSIONS: The ceRNA network of miRNA, lncRNA, and mRNA in peripheral CD19 + B lymphocytes plays an essential role in the pathogenesis of ITP.

Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response.

Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.

Allogenic Hematopoietic Stem Cell Transplant in Iranian Patients With Congenital Sideroblastic Anemia: A Single-Center Experience.

Congenital sideroblastic anemia is characterized by anemia and intramitochondrial iron accumulation in erythroid precursors that form ring sideroblasts. The most common recessive forms are caused by sequence variations in the ALAS2 and SLC25A38 genes. In patients with transfusion-dependent and pyridoxine- resistant severe congenital sideroblastic anemia, hematopoietic stem celltransplantis the only curative option. Herein, we described successful implementations of allogeneic hematopoietic stem cell transplant in 4 Iranian children with congenital sideroblastic anemia. The patients had presented with clinical manifestations of anemia early in life, and the diagnoses of congenital sideroblastic anemia were established through blood tests and bone marrow aspiration. Congenital sideroblastic anemia was further confirmed by the identification of pathogenic variants in SLC25A38 in 2 patients. All 4 patients received allogeneic hematopoietic stem cell transplant with myeloablative conditioning regimen that included busulfan, cyclophosphamide, andrabbit antithymocyte globulin. A combination of cyclosporine A and methotrexate or mycophenolate mofetil was used for graft-versus-host disease prophylaxis. Bone marrow and peripheral blood from sibling or related donors with fully matched human leukocyte antigen profiles were applied. The outcomes of hematopoietic stem celltransplantin patients with congenital sideroblastic anemia were favorable. Three patients achieved full donor chimerism (>95%, 98%, and 100%), and the other patient showed mixed chimerism (75%). All patients remained transfusion independent. Hemato- poietic stem celltransplantis a curative treatmentthat can provide long-term survival for patients with congenital sideroblastic anemia, particularly when used in a timely manner. There remain ongoing challenges in various aspects of hematopoietic stem celltransplantin patients with congenital sideroblastic anemia, which remain to be elucidated.

Dysregulation of Iron Metabolism-Linked Genes at Myocardial Tissue and Cell Levels in Dilated Cardiomyopathy.

In heart failure, the biological and clinical connection between abnormal iron homeostasis, myocardial function, and prognosis is known; however, the expression profiles of iron-linked genes both at myocardial tissue and single-cell level are not well defined. Through publicly available bulk and single-nucleus RNA sequencing (RNA-seq) datasets of left ventricle samples from adult non-failed (NF) and dilated cardiomyopathy (DCM) subjects, we aim to evaluate the altered iron metabolism in a diseased condition, at the whole cardiac tissue and single-cell level. From the bulk RNA-seq data, we found 223 iron-linked genes expressed at the myocardial tissue level and 44 differentially expressed between DCM and NF subjects. At the single-cell level, at least 18 iron-linked expressed genes were significantly regulated in DCM when compared to NF subjects. Specifically, the iron metabolism in DCM cardiomyocytes is altered at several levels, including: (1) imbalance of Fe3+ internalization (SCARA5 down-regulation) and reduction of internal conversion from Fe3+ to Fe2+ (STEAP3 down-regulation), (2) increase of iron consumption to produce hemoglobin (HBA1/2 up-regulation), (3) higher heme synthesis and externalization (ALAS2 and ABCG2 up-regulation), (4) lower cleavage of heme to Fe2+, biliverdin and carbon monoxide (HMOX2 down-regulation), and (5) positive regulation of hepcidin (BMP6 up-regulation).

ALAS1 associated with goat kidding number trait was regulated by the transcription factor ASCL2 to affect granulosa cell proliferation.

ALAS1 is a member of the α-oxoamine synthase family, which is the first rate-limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation-related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real-time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real-time quantitative PCR, EdU and Cell Counting Kit-8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats.

Transcriptome and metabolome analyses reveal the interweaving of immune response and metabolic regulation in pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: The pathogenesis of pelvic organ prolapse (POP) remains unknown. Herein, we aim to reveal the molecular profile of POP by transcriptomic and metabolomic analysis. METHODS: We selected 12 samples of uterosacral ligaments (USLs) from 6 POP patients and 6 controls for transcriptomic and metabolomic analyses. Differentially expressed genes (DEGs) were identified using the R package edgeR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using clusterProfiler, and a protein-protein interaction (PPI) network was constructed using STRING and visualized in Cytoscape. Metabolomic profiling was performed by a liquid chromatography-tandem mass spectrometry system. RESULTS: Transcriptomic analysis identified 487 DEGs between the POP and control groups. Functional enrichment analysis revealed that they were mostly related to immune response terms, including "adaptive immune response," "T cell differentiation," and "T cell activation." In addition, PTPRC, LCK, CD247, IL2RB, CD2, CXR5, JUN, CD3E, IL2RG, and PRF1 were the 10 nodes with the highest node degrees in the PPI network. Metabolomic profiling revealed 290 differentially expressed metabolites, which significantly enriched in "glycerophospholipid metabolism," "nicotinate and nicotinamide metabolism," "glycine, serine, and threonine metabolism," "arginine and proline metabolism," "pyrimidine metabolism," and "purine metabolism." Finally, integrated analysis revealed that the DEGs involved in these significantly enriched metabolic pathways included NT5C1A, GMPR, SDS, ALAS2, CARNS1, PYCR1, P4HA3, PGS1, and NMRK2. CONCLUSIONS: Our findings demonstrate that the immune response and metabolic regulatory pathways are intertwined in POP and might provide new therapeutic targets.

Construction of 5-aminolevulinic acid synthase variants by cysteine-targeted mutation to release heme inhibition.

5-Aminolevulinic acid (5-ALA), a vital precursor for the biosynthesis of tetrapyrrole compounds, has been widely applied in agriculture and medicine, while extremely potential for the treatment of cancers, corona virus disease 2019 (COVID-19) and metabolic diseases in recent years. With the development of metabolic engineering and synthetic biology, the biosynthesis of 5-ALA has attracted increasing attention. 5-Aminolevulinic acid synthase (ALAS), the key enzyme for 5-ALA synthesis in the C4 pathway, is subject to stringent feedback inhibition by heme. In this work, cysteine-targeted mutation of ALAS was proposed to overcome this drawback. ALAS from Rhodopseudomonas palustris (RP-ALAS) and Rhodobacter capsulatus (RC-ALAS) were selected for mutation and eight variants were generated. Variants RP-C132A and RC-C201A increased enzyme activities and released hemin inhibition, respectively, maintaining 82.5% and 81.9% residual activities in the presence of 15 µM hemin. Moreover, the two variants exhibited higher stability than that of their corresponding wild-type enzymes. Corynebacterium glutamicum overexpressing RP-C132A and RC-C201A produced 14.0% and 21.6% higher titers of 5-ALA than the control, respectively. These results strongly suggested that variants RP-C132A and RC-C201A obtained by utilizing cysteine-targeted mutation strategy released hemin inhibition, broadening their applications in 5-ALA biosynthesis.

A Secondary Metabolic Enzyme Functioned as an Evolutionary Seed of a Primary Metabolic Enzyme.

The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an 'evolutionary seed' to generate an essential primary metabolic enzyme.

Congenital sideroblastic anemia model due to ALAS2 mutation is susceptible to ferroptosis.

X-linked sideroblastic anemia (XLSA), the most common form of congenital sideroblastic anemia, is caused by a germline mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. In XLSA, defective heme biosynthesis leads to ring sideroblast formation because of excess mitochondrial iron accumulation. In this study, we introduced ALAS2 missense mutations on human umbilical cord blood-derived erythroblasts; hereafter, we refer to them as XLSA clones. XLSA clones that differentiated into mature erythroblasts showed an increased frequency of ring sideroblast formation with impaired hemoglobin biosynthesis. The expression profiling revealed significant enrichment of genes involved in ferroptosis, which is a form of regulated cell death induced by iron accumulation and lipid peroxidation. Notably, treatment with erastin, a ferroptosis inducer, caused a higher proportion of cell death in XLSA clones. XLSA clones exhibited significantly higher levels of intracellular lipid peroxides and enhanced expression of BACH1, a regulator of iron metabolism and potential accelerator of ferroptosis. In XLSA clones, BACH1 repressed genes involved in iron metabolism and glutathione synthesis. Collectively, defective heme biosynthesis in XLSA clones could confer enhanced BACH1 expression, leading to increased susceptibility to ferroptosis. The results of our study provide important information for the development of novel therapeutic targets for XLSA.

Structural basis for dysregulation of aminolevulinic acid synthase in human disease.

Heme is a critical biomolecule that is synthesized in vivo by several organisms such as plants, animals, and bacteria. Reflecting the importance of this molecule, defects in heme biosynthesis underlie several blood disorders in humans. Aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in α-proteobacteria and nonplant eukaryotes. Debilitating and painful diseases such as X-linked sideroblastic anemia and X-linked protoporphyria can result from one of more than 91 genetic mutations in the human erythroid-specific enzyme ALAS2. This review will focus on recent structure-based insights into human ALAS2 function in health and how it dysfunctions in disease. We will also discuss how certain genetic mutations potentially result in disease-causing structural perturbations. Furthermore, we use thermodynamic and structural information to hypothesize how the mutations affect the human ALAS2 structure and categorize some of the unique human ALAS2 mutations that do not respond to typical treatments, that have paradoxical in vitro activity, or that are highly intolerable to changes. Finally, we will examine where future structure-based insights into the family of ALA synthases are needed to develop additional enzyme therapeutics.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.