Heme Precursor Research Articles

5-Aminolevulinic acid combined with ferrous iron ameliorates myocardial ischemia/reperfusion injury by increasing heme oxygenase-1.

5-Aminolevulinic acid (5-ALA) is a naturally occurring metabolic precursor of heme, and 5-ALA combined with ferrous iron can induce heme oxygenase-1 (HO-1) in various cells. In this study, we investigated the cardioprotective effect of 5-ALA after myocardial ischemia/reperfusion (I/R) injury using a murine model. Male C57BL/6J mice (10-12weeks of age and weighing 21-26g) were pretreated with 100mg/kg of 5-ALA hydrochloride and 157mg/kg of sodium ferrous citrate (SFC) or vehicle 48h, 24h, and 1h before I/R, and underwent 50min of left coronary artery occlusion followed by reperfusion. Infarct area (IA) and area at risk (AAR) were determined by Evans blue and triphenyltetrazolium chloride double staining after reocclusion. Pre-administration with 5-ALA/SFC significantly reduced IA/AAR compared with placebo (34.0% vs. 51.7%, respectively; p = 0.001). Real-time PCR assay after reperfusion showed that mRNA expressions of TNF-α, IL-1β, and BNP were significantly lower, and that of HO-1 was significantly higher in the 5-ALA/SFC group than in the vehicle group in ischemic sites. An inhibition experiment revealed that zinc protoporphyrin IX, an inhibitor of HO-1, inhibited the cardioprotective effects of 5-ALA/SFC. These results suggest that 5-ALA/SFC might play a cardioprotective role in myocardial I/R injury by attenuating the inflammatory reaction by increasing the expression of HO-1.

An adaptor for feedback regulation of heme biosynthesis by the mitochondrial protease CLPXP.

Heme biosynthesis is tightly coordinated such that essential heme functions including oxygen transport, respiration, and catalysis are fully supplied without overproducing toxic heme precursors and depleting cellular iron. The initial heme biosynthetic enzyme, ALA synthase (ALAS), exhibits heme-induced degradation that is dependent on the mitochondrial AAA+ protease complex CLPXP, but the mechanism for this negative feedback regulation had not been elucidated. By biochemical reconstitution, we have discovered that POLDIP2 serves as a heme-sensing adaptor protein to deliver ALAS for degradation. Similarly, loss of POLDIP2 strongly impairs ALAS turnover in cells. POLDIP2 directly recognizes heme-bound ALAS to drive assembly of the degradation complex. The C-terminal element of ALAS, truncation of which leads to a form of porphyria (XLDPP), is dispensable for interaction with POLDIP2 but necessary for degradation. Our findings establish the molecular basis for heme-induced degradation of ALAS by CLPXP, establish POLDIP2 as a substrate adaptor for CLPXP, and provide mechanistic insight into two forms of erythropoietic protoporphyria linked to CLPX and ALAS.

Production of 17α-hydroxyprogesterone using an engineered biocatalyst with efficient electron transfer and improved 5-aminolevulinic acid synthesis coupled with a P450 hydroxylase

17α-Hydroxyprogesterone (17α-OH-PROG) is an important intermediate with a wide range of applications in the pharmaceutical industry. Strategies based on efficient electron transfer and cofactor regeneration were used for the production of 17α-OH-PROG. Here, CYP260A1, Fpr and Adx were expressed using a double plasmid system, resulting in higher biotransformation efficiency. Further optimization of reaction conditions and addition of polymyxin B increased the production of 17α-OH-PROG from 12.52 mg/L to 102.37 mg/L after 12 h of biotransformation. To avoid the addition of external 5-aminolevulinic acid (ALA) as a heme precursor for the P450 enzyme, a modified C5 pathway was introduced into the engineered strain, further reducing the overall process cost. The resulting whole-cell biocatalyst achieved the highest biotransformation yield of 17α-OH-PROG reported to date, offering a promising strategy for commercial application of P450 enzymes in industrial production of hydroxylated intermediates.

Effects of iron supplements in individuals with erythropoietic protoporphyria

BackgroundProtoporphyrin IX (PPIX) is the final precursor of heme, forming heme when iron is inserted. Individuals with erythropoietic protoporphyrias (EPP) have accumulation of PPIX, causing photosensitivity and increased liver disease risk. Many also have iron deficiency and anemia. We investigated outcomes of oral iron supplements in individuals with EPP. MethodsA systematic review identified literature on oral iron supplements in EPP patients. Subsequently, we administered iron supplements to EPP patients with iron deficiency. The primary outcome was impact on PPIX level. Secondary outcomes were adverse events and relative differences in hemoglobin and iron parameters. ResultsThe systematic review found 13 case reports and one uncontrolled clinical trial with uncertain results. From our department 10 patients with EPP and iron deficiency took daily dosages of 330 mg of ferrous fumarate for two months. Five of our patients had anemia at baseline. After 2 months of supplementation seven patients had increased PPIX level compared to baseline, two had decrease, one remained unchanged. The administration of iron led to a rise in ferritin, and in four of the anemic patients also to an improvement in blood hemoglobin. A small transiently elevation in plasma alanine transaminase concentration was observed during supplementation. ConclusionsOverall, iron supplementation in EPP patients replenished iron stores and elevated erythrocyte PPIX and plasma alanine transaminase. For anemic patients, there was some degree of normalization of the hemoglobin level. If iron therapy is needed for EPP patients, monitoring of photosensitivity, PPIX, hemoglobin, and plasma liver enzymes is advisable.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

State of the Art Technologies for High Yield Heterologous Expression and Production of Oxidoreductase Enzymes: Glucose Oxidase, Cellobiose Dehydrogenase, Horseradish Peroxidase, and Laccases in Yeasts P. pastoris and S. cerevisiae

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in the food industry and cosmetics (glucose oxidase (GOx) and cellobiose dehydrogenase (CDH)), bioremediations (horseradish peroxidase (HRP) and laccase (LAC)), and medicine for biosensors and miniature biofuel cells (GOx, CDH, LAC, and HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with the heterologous expression of oxidoreductase enzymes, since there is a requirement not only for multiple OXR gene integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition and fermentation conditions during expression due to the need for transition metals (copper and iron) and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find the optimal formula using the above-mentioned approaches; most recently, researcher started using protein engineering and directed evolution to increase in the yield of recombinant enzyme production. In this review article, we will cover all the current state-of-the-art technologies and most recent advances in the field that yielded a high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increases in fermentation yield using cutting-edge technologies such as directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry, and microfluidics.

High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway.

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO4·7H2O, a heme content of 66.32mg/L and an intracellular LBA titer of 7.27g/L were achieved in the engineered strain in a 5L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

Application of a Validated Method for Quantifying Circulating Protoporphyrin IX to the Beacon Trial of Bitopertin in Erythropoietic Protoporphyria

Introduction: Accumulation of the photoreactive heme precursor protoporphyrin IX (PPIX) is a signature of the disease pathophysiology for both erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP), and PPIX levels correlate with disease severity and light tolerance. Additionally, an elevated ratio of circulating metal-free PPIX vs. metal-bound PPIX is observed in patients with protoporphyrias, and the extent of metal-free elevation is distinguishable between EPP (85-100%) and XLP (50-85%). Therefore, the accurate determination of metal-free PPIX and metal-bound PPIX is of particular interest in patients with EPP or XLP. Methods: Novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed and validated to quantify circulating metal-free and zinc (Zn)-complexed PPIX in whole blood and plasma. Each of the four assays were developed and validated according to applicable regulatory guidance for bioanalytical assay validation with regard to accuracy, precision, sensitivity, and selectivity. The minimum required sample volume for each assay was 200 µL, compared to >1 mL for most currently available protoporphyria-related assays. During bioanalytical method development, rapid degradation of metal-free PPIX (≥90%) and Zn-PPIX (up to 96%) was observed in plasma samples within 24 hours under ambient light conditions. In contrast, whole blood was less susceptible to photodegradation (i.e., ≤20% for both metal-free PPIX and Zn-PPIX) under the same conditions. The most effective method for maintaining sample integrity was reduction of light exposure during sample collection, handling, and processing. Implementation of light-reduction controls was used during method validation and facilitated improved stability of metal-free PPIX and Zn-PPIX in plasma and whole-blood samples. For the metal-free PPIX plasma assay validation, the effect of hemolysis was evaluated due to the apparent sequestration of metal-free PPIX in red blood cells. The bioanalytical work informed specific sample-collection procedures to mitigate instability and hemolysis, such as the use of opaque consumables for sample collection, controls for plasma aliquoting, and operating under low-light conditions for all sample handling and processing. BEACON is a Phase 2, randomized, open-label, parallel-arm trial (ACTRN12622000799752) of approximately 22 participants who will receive oral, once-daily administration of 20 or 60 mg of bitopertin for 6 months. Baseline light tolerance was assessed using 2 patient-reported outcomes measures during a 2- to 4-week screening period. Participants were asked to conduct weekly sun exposure challenges and time to first prodromal symptom (itching, burning, tingling, etc.) was averaged for the screening period. Results: More than 600 clinical samples from 17 participants in the BEACON study have been collected as of 06 June 2023 and analyzed using the validated porphyrin assays. Measured metal-free PPIX baseline values ranged from 1440 - 34,100 ng/mL (2.6 - 61 µM) in whole blood and 177 - 2560 ng/mL (0.31 - 4.5 µM) in plasma ( n=16). Baseline percentage of intra-patient plasma/whole-blood concentrations ranged from 2.5 - 16%. Each participant's baseline values were converted to erythrocyte metal-free PPIX concentrations using measured hematocrit levels and ranged from 3590 - 88400 ng/mL (6.4 - 157 µM). These values were comparable to data previously reported in the literature (6-139 µM 1). Additionally, higher baseline whole-blood metal-free PPIX concentrations were associated with decreased averages for time to prodrome (Table 1). Conclusion: Fully-validated LC-MS/MS assays to quantify metal-free PPIX and Zn-PPIX in plasma and whole blood were used to analyze clinical samples from the BEACON study evaluating bitopertin in EPP. PPIX values were consistent with previously reported values for patients with EPP. The methods also provided improved accuracy and specificity for metal-free vs. metal-complexed PPIX and reduced sample volume requirements. In addition, higher baseline whole-blood PPIX values were associated with decreased light tolerance within the trial population, supporting its use as a measure of disease severity in EPP.

A role of splenic heme biosynthesis pathway in the persistent prophylactic actions of arketamine in lipopolysaccharide-treated mice

Relapse is common in remitted patients with major depressive disorder (MDD). Arketamine, an (R)-enantiomer of ketamine, has persistent prophylactic actions in an inflammatory model of depression. However, the precise mechanisms underlying these prophylactic actions remain unknown. Given the role of the brain–spleen axis in depression, we sought to identify splenic molecular targets that play a role in the prophylactic actions of arketamine. Lipopolysaccharide (LPS) (1.0 mg/kg) was administered 6 days after a single injection of arketamine (10 mg/kg) or saline. RNA-sequencing analysis found altered expression in the heme biosynthesis II pathway. Quantitative RT-PCR revealed that pretreatment with arketamine blocked increased expression of genes involved in the heme biosynthesis II pathway in LPS-treated mice, namely, 5-aminolevulinase synthase 2 (Alas2), ferrochelatase (Fech), hydroxymethylbilane synthase (Hmbs). Interestingly, there were positive correlations between the expression of these genes and spleen weight or plasma levels of pro-inflammatory cytokines. We also found higher expression of ALAS2 and FECH in the spleen from MDD patients. Pretreatment with a key intermediate precursor of heme, 5-aminolaevulinic acid (300 mg/kg/day for 3 days), caused splenomegaly, higher plasma levels of pro-inflammatory cytokines, and depression-like behavior in low-dose LPS (0.1 mg/kg)-treated mice. Interestingly, pretreatment with a heme biosynthesis inhibitor, succinyl acetone (120 mg/kg/day for 3 days), had prophylactic effects in LPS (1.0 mg/kg)-treated mice. These data suggest a novel role for the heme biosynthesis II pathway in the spleen for inflammation-related depression. Therefore, the heme biosynthesis pathway could be a new target for the prevention of relapse in MDD patients.

5-Aminolevulinic acid bypasses mitochondrial complex I deficiency and corrects physiological dysfunctions in Drosophila.

Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.

5‑aminiolevulinic acid induces a radiodynamic effect with enhanced delayed reactive oxygen species production under hypoxic conditions in lymphoma cells: An invitro study.

Primary central nervous system lymphoma (PCNSL) is a rare and aggressive type of intracranial tumor. However, PCNSL is radiosensitive; thus, whole-brain radiotherapy (WBRT) is often selected as an alternative consolidation therapy. WBRT-related delayed neurotoxicity can affect the quality of life of the elderly. 5-aminolevulinic acid (ALA) is a natural precursor of heme and has been widely used as a live molecular fluorescence marker in brain tumor surgery. Experimental studies have demonstrated that combination therapy with 5-ALA and ionizing irradiation (IR), denoted radiodynamic therapy (RDT), resulted in tumor suppression in cancer, including glioma, melanoma, colorectal cancer, prostate cancer, breast cancer and lung cancer; however, to the best of our knowledge, this method has not been investigated in lymphoma. The present study aimed to investigate the radiodynamic effect of 5-ALA on lymphoma cells in vitro. The synthesis of 5-ALA-induced protoporphyrin IX (PpIX) was assessed under normal and hypoxic conditions in lymphoma cells (Raji, HKBML and TK). Subsequently, the radiodynamic effect of 5-ALA was evaluated using a colony formation assay and reactive oxygen species (ROS) production after RDT was examined using flow cytometry. Finally, the mitochondrial density in the lymphoma cells was evaluated. Lymphoma cells exhibited a high accumulation of 5-ALA-induced PpIX in the flow cytometric analysis, and a decrease in the surviving fraction under IR in cells with 5-ALA treatment compared with cells not treated with 5-ALA in the colony formation assay under normal and hypoxic conditions. Although ROS production 12 h after IR was increased compared with that immediately after IR (0 h), pretreatment with 5-ALA enhanced the delayed ROS production in each lymphoma cell line under normoxic conditions. Raji and TK cells exhibited an increase in ROS production 12 h after IR compared with that at 0 h in the 5-ALA-untreated cells under hypoxic conditions. Raji, HKBML and TK cells exhibited an increase in ROS production 12 h after IR compared with that at 0 h in the 5-ALA-treated cells, while TK cells exhibited enhancement of ROS production 12 h after IR in 5-ALA-treated cells compared with 5-ALA-untreated cells under hypoxic conditions. Other studies have demonstrated that impaired mitochondria damaged by IR produce ROS via the metabolic process, then damage the rest of the surrounding normal mitochondria, consequently propagating oxidative stress within tumor cells and leading to cell death. Thus, we hypothesized that the propagating oxidative stress after IR was associated with mitochondrial density in tumor cells. Namely, high accumulation of 5-ALA-indcued PpIX may promote ROS production in mitochondria of tumor cells after IR, and suppress the cell surviving fraction via the propagation of oxidative stress. In the colony formation assay, Raji cell colony formation was suppressed by RDT with 5-ALA. Simultaneously, the mitochondrial density in the Raji cells was higher than that in other cell lines. Pretreatment with 5-ALA enhanced delayed ROS production after IR in lymphoma cells under normoxic conditions. Under hypoxic conditions, only TK cells exhibited enhancement of ROS production 12 h after IR in the 5-ALA-treated group compared with the 5-ALA-untreated group. Although further studies evaluating the effect of hypoxic conditions in lymphoma cells are needed, the results suggested that RDT with 5-ALA could suppress colony formation under normal and hypoxic conditions in lymphoma cells. Therefore, RDT with 5-ALA is a potential treatment option for PCNSL.

Heme biosensor-guided in vivo pathway optimization and directed evolution for efficient biosynthesis of heme

BackgroundHeme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe2+ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis.ResultsWe report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme.ConclusionWe sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.

Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

Understanding Carbohydrate Metabolism and Insulin Resistance in Acute Intermittent Porphyria.

Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks associated with high production, accumulation and urinary excretion of heme precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). The estimated clinical penetrance for AIP is extremely low (<1%), therefore it is likely that other factors may play an important role in the predisposition to developing attacks. Fasting is a known triggering factor. Given the increased prevalence of insulin resistance in patients and the large urinary loss of succinyl-CoA to produce ALA and PBG, we explore the impact of reduced availability of energy metabolites in the severity of AIP pathophysiology. Classic studies found clinical improvement in patients affected by AIP associated with the administration of glucose and concomitant insulin secretion, or after hyperinsulinemia associated with diabetes. Molecular studies have confirmed that glucose and insulin administration induces a repressive effect on hepatic ALA Synthase, the first and regulatory step of the heme pathway. More recently, the insulin-mimicking α-lipoic acid has been shown to improve glucose metabolism and mitochondrial dysfunction in a hepatocyte cell line transfected with interfering RNA targeting PBGD. In AIP mice, preventive treatment with an experimental fusion protein of insulin and apolipoprotein A-I improved the disease by promoting fat mobilization in adipose tissue, increasing the metabolite bioavailability for the TCA cycle and inducing mitochondrial biogenesis in the liver. In this review, we analyze the possible mechanisms underlying abnormal hepatocellular carbohydrate homeostasis in AIP.

Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

Adverse Effect of Blue Light on DNA Integrity Is Accelerated by 5-Aminolevulinic Acid in HaCaT Human Keratinocyte Cells and B16F1 Murine Melanoma Cells.

Several studies have suggested the potential benefits of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a precursor of heme, which generates reactive oxygen species (ROS) following photoirradiation. Some reports indicate that blue light induces intracellular ROS production. In the present study, we elucidated the effects of blue light and 5-ALA on DNA integrity in B16F1 murine melanoma and human keratinocyte HaCaT cells using a variety of comet assay techniques. Co-treatment with blue light and 5-ALA significantly decreased cell viability in both cell lines. A neutral comet assay was performed to assess DNA double-strand break (DSB) formation and blue light and 5-ALA caused DSBs. We also performed an alkali comet assay to detect single-strand breaks (SSB) and alkali labile sites (ALS). The results indicated that 5-ALA accelerated blue light-induced SSB formation. In addition, modified comet assays were done using two types of enzymes to evaluate oxidative DNA damages. The results indicated that blue light and 5-ALA generated oxidized purine and pyrimidines in both cell lines. In summary, co-treatment with 5-ALA and photoirradiation may cause unexpected DNA damage in cells and tissues.

Protoporphyrin IX derived from dual-species anaerobic biofilms of Fusobacterium necrophorum and Porphyromonas levii attenuates bovine neutrophil function

Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm-bacteria in modulating host immune function is less well understood. A model of mixed-species biofilms composed of Fusobacterium necrophorum and Porphyromonas levii was developed to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that different soluble extracellular factors (ECFs) would be released from planktonic and biofilm bacteria, resulting in altered neutrophil function. Neutrophils exposed to ECFs from planktonic bacteria showed significantly elevated levels of reactive oxygen species (ROS). In contrast, biofilm components from these same species of bacteria failed to induce such a response. Size-exclusion filtration of ECFs revealed that the bioactive molecule causing neutrophil ROS responses was below 3 kDa. Intensive heat, nuclease, lipase, or protease treatments of the <3 kDa fractions did not alter neutrophil functional responses. Protoporphyrin IX (PPIX) is an important heme precursor and growth requirement for many anaerobes. Porphyromonas species can accumulate environmental PPIX at the cell surface as a strategy to protect the bacteria from oxidative stress and we investigated the direct interaction of bovine neutrophils with PPIX. In the present study, evidence suggests that the accumulation of protoporphyrin in these dual-species biofilm ECFs attenuates neutrophil ROS production and chemotaxis. The diminished neutrophil response to biofilm ECFs via the action of PPIX may represent a biofilm immune-evasion strategy that could assist in explaining the ineffectual host clearance of biofilm-mediated infections involving these bacteria.

Conditional Synthetic Lethality between the Initial Steps of Heme Biosynthesis and Pyridoxine Metabolism during Erythropoiesis

Heme biosynthesis depends upon pyridoxal 5' phosphate (PLP), the active form of pyridoxine or vitamin B6. PLP is the cofactor of ALAS, the first enzyme of heme biosynthesis that condenses glycine and succinyl-CoA to form 5-ALA, the sole precursor of porphyrins and heme. PLP is also the cofactor for multiple other enzymes relevant to heme synthesis, notably several involved in glycine metabolism. ALAS2 is the erythroid-specific isoform of ALAS. Loss-of-function and gain-of-function mutations in ALAS2 cause two rare diseases: X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP), respectively. We recently generated mouse models of XLSA and XLPP harboring knock-in mutations found in patients; these mouse models recapitulate their respective human diseases. Using diets with defined amounts of pyridoxine, we demonstrated that XLSA mutant animals of three different genotypes (R170H, R411H, and R452H), are dependent upon pyridoxine to ensure their erythropoiesis and red blood cells (RBC) formation, independent of the initial severity of the anemia under baseline conditions. Control littermates or XLPP animals only develop moderate sideroblastic anemia (hemoglobin >8g/dL) after eight weeks of a pyridoxine-deficient diet, which corresponds roughly to RBC lifespans. Whereas pyridoxine-deficient XLSA animals develop massive splenomegaly and a progressively profound anemia in the same period. Methodology. To determine if this phenomenon is specific to XLSA, we assessed the effect of pyridoxine deficiency in other mouse models of erythroid heme or globin synthesis. We used an erythroid protoporphyria (EPP) model (Fechm1Pas/m1Pas), a beta-thalassemia model (HBBTh3/+), and a new model of congenital sideroblastic anemia (SLC25A38c/-vav-cre+). SLC25A38 is the only known mitochondrial importer of glycine, and the anemia of SLC25A38-CSA patients is typically pyridoxine non-responsive. At baseline, SLC25A38c/-vav-cre+ animals have a moderate microcytic hypochromic anemia with abundant siderocytes. For each of these strains, we fed male animals diets containing 0, 10, or 100 ppm pyridoxine at weaning. Complete blood counts (2, 5, and 8 weeks of diet) and steady-state and stress erythropoiesis (8 weeks of diet) were assessed. Results. Pyridoxine-depleted EPP animals developed an anemia similar to the control and XLPP animals but continued to accumulate free protoporphyrin in RBCs. The anemia in beta-thalassemia animals was not substantially impacted by pyridoxine deficiency. In contrast, the response of SLC25A38c/- vav-cre+ mice to pyridoxine deficiency was dramatic. After only three weeks of depletion, the anemia was already severe (HGB <2g/dL). In pyridoxine-depleted animals, the remaining RBCs did not present with siderocytes. Notably, the spleen size was normal, and no blockage of erythropoiesis was found by flow cytometry. Instead, both the marrow and spleen contained <15% ter119+ cells, half of them being RBCs, suggesting a profound and early disruption of erythropoiesis. Conclusion. Heme-dependent Congenital Sideroblastic Anemia (heme-CSA), specifically XLSA and SLC25A38-CSA, and pyridoxine deficiency are non-lethal in mouse models. Nevertheless, heme-CSA mouse models are not viable in the absence of pyridoxine due to impaired erythropoiesis. This phenomenon can be considered conditional synthetic lethality between early heme biosynthesis and pyridoxine metabolism in erythroid cells. Furthermore, an additional PLP-dependent mechanism in both diseases supports the impaired erythropoiesis induced by the genetic mutation. As PLP is not only ALAS2 cofactor, including many enzymes involved in glycine metabolism, which contribute to heme synthesis, identifying the underlying PLP pathway(s) that compensate for other enzymatic defects could offer new therapeutic approaches for heme-CSAs.

Interaction of protoporphyrinix (PPIX) and 5-amino levulinic acid (ALA) in nanoemulsion

Nanoemulsion is an interesting and unique fluid system in that it is used to solubilize both ionic and non-ionic molecules. Because of its uniqueness it has been used as a medium for drug delivery. It is therefore used in this work to study the interaction of Protoporphyrin (PPIX) and 5-Amino levulinic acid (ALA). While ALA is a distant precursor of PPIX, PPIX itself is not only a precursor of Heme but also a photosensitizer in the modality of Photodynamic Therapy (PDT). Both compounds are used use in PDT regimen. A steady-state fluorescence technique is used for the study of the interaction of these very important biological compounds. It is found that ALA quenches the fluorescence of PP IX in nanoemulsion. This observed quenching is diffusion controlled. The bimolecular quenching constant, kq, was determined as 2.86x 1010/M-s with an interaction constant, Ka, of 4.48 x 105 with the free energy of interaction, ΔGa of -32.234kJ/mole.

Iron Metabolism in the Disorders of Heme Biosynthesis.

Given its remarkable property to easily switch between different oxidative states, iron is essential in countless cellular functions which involve redox reactions. At the same time, uncontrolled interactions between iron and its surrounding milieu may be damaging to cells and tissues. Heme—the iron-chelated form of protoporphyrin IX—is a macrocyclic tetrapyrrole and a coordination complex for diatomic gases, accurately engineered by evolution to exploit the catalytic, oxygen-binding, and oxidoreductive properties of iron while minimizing its damaging effects on tissues. The majority of the body production of heme is ultimately incorporated into hemoglobin within mature erythrocytes; thus, regulation of heme biosynthesis by iron is central in erythropoiesis. Additionally, heme is a cofactor in several metabolic pathways, which can be modulated by iron-dependent signals as well. Impairment in some steps of the pathway of heme biosynthesis is the main pathogenetic mechanism of two groups of diseases collectively known as porphyrias and congenital sideroblastic anemias. In porphyrias, according to the specific enzyme involved, heme precursors accumulate up to the enzyme stop in disease-specific patterns and organs. Therefore, different porphyrias manifest themselves under strikingly different clinical pictures. In congenital sideroblastic anemias, instead, an altered utilization of mitochondrial iron by erythroid precursors leads to mitochondrial iron overload and an accumulation of ring sideroblasts in the bone marrow. In line with the complexity of the processes involved, the role of iron in these conditions is then multifarious. This review aims to summarise the most important lines of evidence concerning the interplay between iron and heme metabolism, as well as the clinical and experimental aspects of the role of iron in inherited conditions of altered heme biosynthesis.