Heme Biosynthesis Research Articles

Data-independent acquisition-based blood proteomics unveils predictive biomarkers for neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a life-threatening condition affecting preterm infants, sometimes necessitating surgical treatment. This study aimed to analyze differentially expressed proteins (DEPs) and access their biological and clinical significance in the plasma of neonates with NEC. Peripheral blood samples were collected from NEC infants at various time points, and plasma was separated. Data-independent acquisition (DIA) technology was utilized to identify DEPs among NEC patients at different stages. Bioinformatic analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, and protein-to-protein interaction analyses were performed on the DEPs. External datasets, along with receiver operating characteristic curves and gene set enrichment analysis, were used to clinically and biologically validate the findings. DEPs between the NEC and pre-NEC groups indicated reduced protein, heme, nitrogen, and purine nucleotide biosynthesis during NEC formation. In addition, enriched DEPs among the NEC groups at different time points suggested reconstructed extracellular matrix, aberrant B-lymphocyte immune responses, and decreased glycosaminoglycan levelsduring NEC progression. These findings were both clinically and biologically validated using external datasets. Our study highlights the clinical and biological relevance of proteomics in NEC patients. This study demonstrates key pathways involved in NEC pathogenesis and establishes DIA mass spectrometry as a powerful and noninvasive tool for evaluating and predicting NEC formation and progression.

A glutamine metabolic switch supports erythropoiesis.

Metabolic requirements vary during development, and our understanding of how metabolic activity influences cell specialization is incomplete. Here, we describe a switch from glutamine catabolism to synthesis required for erythroid cell maturation. Glutamine synthetase (GS), one of the oldest functioning genes in evolution, is activated during erythroid maturation to detoxify ammonium generated from heme biosynthesis, which is up-regulated to support hemoglobin production. Loss of GS in mouse erythroid precursors caused ammonium accumulation and oxidative stress, impairing erythroid maturation and recovery from anemia. In β-thalassemia, GS activity is inhibited by protein oxidation, leading to glutamate and ammonium accumulation, whereas enhancing GS activity alleviates the metabolic and pathological defects. Our findings identify an evolutionarily conserved metabolic adaptation that could potentially be leveraged to treat common red blood cell disorders.

Herbicides as fungicides: Targeting heme biosynthesis in the maize pathogen Ustilago maydis.

Pathogens must efficiently acquire nutrients from host tissue to proliferate, and strategies to block pathogen access therefore hold promise for disease control. In this study, we investigated whether heme biosynthesis is an effective target for ablating the virulence of the phytopathogenic fungus Ustilago maydis on maize plants. We first constructed conditional heme auxotrophs of the fungus by placing the heme biosynthesis gene hem12 encoding uroporphyrinogen decarboxylase (Urod) under the control of nitrogen or carbon source-regulated promoters. These strains were heme auxotrophs under non-permissive conditions and unable to cause disease in maize seedlings, thus demonstrating the inability of the fungus to acquire sufficient heme from host tissue to support proliferation. Subsequent experiments characterized the role of endocytosis in heme uptake, the susceptibility of the fungus to heme toxicity as well as the transcriptional response to exogenous heme. The latter RNA-seq experiments identified a candidate ABC transporter with a role in the response to heme and xenobiotics. Given the importance of heme biosynthesis for U. maydis pathogenesis, we tested the ability of the well-characterized herbicide BroadStar to influence disease. This herbicide contains the active ingredient flumioxazin, an inhibitor of Hem14 in the heme biosynthesis pathway, and we found that it was an effective antifungal agent for blocking disease in maize. Thus, repurposing herbicides for which resistant plants are available may be an effective strategy to control pathogens and achieve crop protection.

Myocardial injury and heart failure biomarkers in acute intermittent porphyria

Abstract Background Acute intermittent porphyria (AIP) is a rare metabolic disorder caused by the defect in the hydroxymethylbilane synthase gene, encoding the third enzyme in heme biosynthesis pathway. The main symptoms include abdominal pain, vomiting, paralysis, confusion. Exacerbation of AIP may manifest with elevation of blood pressure and tachycardia due to hyperactivity of sympathetic nervous system. Data regarding cardiac function in AIP are scarce. Purpose The aim of this study was to assess the myocardial injury and heart failure biomarkers during AIP exacerbations and remission. Methods This cohort prospective study encompassed patients at the age 18-65 years who had at least one exacerbation of AIP in their lifetime. Patients examined during the AIP exacerbations were reassessed in the remission phase. The concentrations of sodium, troponin T, CK-MB mass, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and metoxycatecholamines in blood were assessed. Blood pressure was measured with 24-hour ABPM. Echocardiography was performed according to the current recommendations. Results The total number of 70 patients with AIP were enrolled to the study (April 2019 - March 2023). We assessed 36 patients in both phases of disease, mean age 37 years (21-63), 75% females. The median of prior AIP exacerbations was 8. During the exacerbations a significant increase in the concentrations of NT-proBNP (250 [97 ; 501] vs. 89 [48 ; 140] pg/mL, p<0.001), metanephrine (49.0 [37.8 ; 61.2] vs. 41.4 [36.8 ; 53.3] pg/mL, p=0.009) and normetanephrine (169.8 [118.2 ; 327.2] vs. 93.2 [78.1 ; 120.9] pg/mL, p<0.001) was observed. Conversely, sodium concentrations decreased (136 [133 ; 140] vs. 140 [139 ; 142] mmol/L, p<0.001). There were no significant differences in the values of troponin (5.4 [4.0 ; 11.3] vs. 5.6 [3.7 ; 9.5] ng/L, p=0.445), CK-MB mass (1.88 [0.84 ; 2.64] vs. 1.21 [0.83 ; 2.80] ng/mL, p=0.80). In the acute phases of AIP the levels of NT-proBNP were correlated negatively with the values of sodium concentrations (r=-0.36, p=0.032) and positively with average systolic blood pressure (r=0.32, p=0.06). Echocardiography showed an increase in left ventricular (LV) mass indices in some patients hospitalized due to AIP symptoms. Sodium concentrations correlated negatively with these changes (r=-0.36, p=0.032). LV ejection fraction was slightly higher during AIP exacerbations. There were no significant differences in other parameters of LV systolic and diastolic function. Conclusions During acute intermittent porphyria (AIP) exacerbations there is an increase in the concentrations of NT-proBNP and catecholamines in blood. In most patients the levels of markers of myocardial injury, such as troponin and CK-MB mass, do not differ significantly between phases of disease. During AIP exacerbations some patients experience an increase in LV mass which may be associated with hyponatremia. In most cases cardiac function do not change significantly.Frequency of abnormal laboratory resultsResults of echocardiography

Protoporphyrin IX-Dependent Antiviral Effects of 5-Aminolevulinic Acid against Feline Coronavirus Type II.

5-Aminolevulinic acid (5-ALA), a non-proteinogenic amino acid, is an intermediate in the biosynthesis of heme and exerts antiviral effects against feline coronavirus (FCoV); however, the underlying mechanisms remain unclear. In the biosynthesis of heme, 5-ALA is condensed and converted to protoporphyrin IX (PpIX), which is then transformed into heme by the insertion of ferrous iron. Previous research has suggested that the metabolites generated during heme biosynthesis contribute to the antiviral effects of 5-ALA. Therefore, the present study investigated the in vitro mechanisms responsible for the antiviral effects of 5-ALA. The results obtained revealed that 5-ALA and PpIX both effectively reduced the viral titer in the supernatant of FCoV-infected fcwf-4 cells. Moreover, PpIX exerted virucidal effects against FCoV. We also confirmed that 5-ALA increased PpIX levels in cells. While hemin induced heme oxygenase-1 gene expression, it did not reduce the viral titer in the supernatant. Sodium ferrous citrate decreased PpIX levels and suppressed the antiviral effects of 5-ALA. Collectively, these results suggest that the antiviral effects of 5-ALA against FCoV are dependent on PpIX.

Gene expression profiling in Venous thromboembolism: Insights from publicly available datasets

BackgroundVenous thromboembolism (VTE) is the third most common cardiovascular disease and is a major cause of mobility and mortality worldwide. VTE is a complex multifactorial disease and genetic mechanisms underlying its pathogenesis is yet to be completely elucidated. The aim of the present study was to identify hub genes and pathways involved in development and progression of blood clot during VTE using gene expression data from public repositories. MethodologyDifferential gene expression (DEG) data from two datasets, GSE48000 and GSE19151 were analysed using GEO2R tool. Gene expression data of VTE patients were compared to that of healthy controls using various bioinformatics tools. ResultsWhen the differentially expressed genes of the two datasets were compared, it was found that 19 genes were up-regulated while 134 genes were down-regulated. Gene ontology (GO) and pathway analysis revealed that pathways such as complement and coagulation cascade and B-cell receptor signalling along with DNA methylation, DNA alkylation and inflammatory genes were significantly up-regulated in VTE patients. On the other hand, differentially down-regulated genes included mitochondrial translation elongation, termination and biosysthesis along with heme biosynthesis, erythrocyte differentiation and homeostasis. The top 5 up-regulated hub genes obtained by protein-protein interaction (PPI) network analysis included MYC, FOS, SGK1, CR2 and CXCR4, whereas the top 5 down-regulated hub genes included MRPL13, MRPL3, MRPL11, RPS29 and RPL9. The up-regulated hub genes are functionally involved in maintain vascular integrity and complementation cascade while the down-regulated hub genes were mostly mitochondrial ribosomal proteins. ConclusionPresent study highlights significantly enriched pathways and genes associated with VTE development and prognosis. The data hereby obtained could be used for designing newer diagnostic and therapeutic tools for VTE management.

Functional diversification within the heme-binding split-barrel family

Due to neofunctionalization, a single fold can be identified in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experimentation to reveal a large multi-functional family of putative enzymatic and non-enzymatic proteins involved in heme metabolism. This family (homolog of HugZ (HOZ)) is embedded in the “FMN-binding split barrel” superfamily and contains separate groups of proteins from prokaryotes, plants, and algae, which bind heme and either catalyze its degradation or function as non-enzymatic heme sensors. In prokaryotes these proteins are often involved in iron assimilation, whereas several plant and algal homologs are predicted to degrade heme in the plastid or regulate heme biosynthesis. In the plant Arabidopsis thaliana, which contains two HOZ subfamilies that can degrade heme in vitro (HOZ1 and HOZ2), disruption of AtHOZ1 (AT3G03890) or AtHOZ2A (AT1G51560) causes developmental delays, pointing to important biological roles in the plastid. In the tree Populus trichocarpa, a recent duplication event of a HOZ1 ancestor has resulted in localization of a paralog to the cytosol. Structural characterization of this cytosolic paralog and comparison to published homologous structures suggests conservation of heme-binding sites. This study unifies our understanding of the sequence-structure-function relationships within this multi-lineage family of heme-binding proteins and presents new molecular players in plant and bacterial heme metabolism.

Biosynthesis of plant-derived triterpenoid asiatic acid in Saccharomyces cerevisiae cell factories

Asiatic acid, a bioactive component of Centella asiatica (L.) Urban, exhibits plentiful valuable pharmacological properties. Herein, we engineered Saccharomyces cerevisiae to produce asiatic acid. Initially, asiatic acid was synthesized by expressing the Centella asiatica cytochrome P450 monooxygenases CYP714E19 and CYP716C11 in a Saccharomyces cerevisiae strain optimized for ursolic acid production. The engineered strain yielded 0.42 ± 0.01 mg/L and 0.067 ± 0.0013 mg/g dry cell weight (DCW) of asiatic acid. Subsequently, a suitable cytochrome P450 reductase was screened, and key enzymes were overexpressed to effectively convert ursolic acid to asiatic acid. Strengthening heme biosynthesis, promoting endoplasmic reticulum (ER) expansion, and enhancing the cofactor supply were implemented to improve P450 catalytic activity. Additionally, a PDZ-PDZlig-mediated protein self-assembly strategy was used to improve the efficiency of the CYP714E19 and CYP716C11 catalytic cascade. Finally, the highest production was achieved (30.09 ± 0.15 mg/L, 4.09 ± 0.01 mg/g DCW) in microbial cell factories. This work establishes a foundation for efficient production of asiatic acid.

Comparative study of oxidative stress in cancer patients occupationally exposed to the mixture of pesticides.

Cancer isa leading cause of death worldwide, accounting for nearly 10 million deaths in 2020.Reviews indicated a positive relationship between exposure to pesticides and the development of cancers. In the present study, we have estimated the level of oxidative stress markers in serum samples of pesticide exposure and unexposed cancer patients as compared to normal control. We have found a significant decrease in peroxygenase (PON) and arylesterase (ARE) activity and substantial increases in homocysteine levels in both cancer groups. The level of heme biosynthesis rate-limiting enzymesdelta-aminolevulinic acid dehydratase(δ-ALA-D)also significantly decreased compared to control. The statistical comparison between the cancer groups does not show significant changes. We concluded the involvement of oxidative stress in carcinogenesis in both cancer group patients. However, more study is needed to put homocysteineas a novel marker for a variety of diseases on a single platform.

Systemic messenger RNA replacement therapy is effective in a novel clinically relevant model of acute intermittent porphyria developed in non-human primates

ObjectiveAcute intermittent porphyria (AIP) is a rare metabolic disorder caused by haploinsufficiency of hepatic porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthesis. Individuals with AIP experience neurovisceral attacks...

Metabolic Engineering of Saccharomyces cerevisiae for Fermentative Production of Heme.

Heme is a key ingredient required to mimic the color and flavor of meat in plant-based alternatives. This study aimed to develop a yeast-based microbial cell factory for efficient and sustainable production of heme. To this end, first, Hem12p (uroporphyrinogen decarboxylase) was identified as the rate-limiting enzyme in the heme biosynthetic pathway present in Saccharomyces cerevisiae D452-2. Next, we investigated the effects of disruption of the genes involved in the competition for heme biosynthesis precursors, transcriptional repression, and heme degradation (HMX1) on heme production efficiency. Of the knock-out strains constructed in this study, only the HMX1-deficient strain produced heme at a higher concentration than the background strain without gene disruption. In addition, overexpression of PUG1 encoding a plasma membrane transporter involved in protoporphyrin IX (the precursor to heme biosynthesis) uptake led to a significant increase in intracellular heme concentration. As a result, among the various engineered strains constructed in this study, the ΔHMX1/H3&12+PUG1 strain, the HMX1-deficient strain overexpressing HEM3, HEM12, and PUG1, produced the highest concentration of heme (4.6mg/L) in batch fermentation, which was 3.9-fold higher than that produced by the wild-type D452-2 strain. In a glucose-limited fed-batch fermentation, the ΔHMX1/H3&12+PUG1 strain produced 28mg/L heme in 66h.

Heme deficiency in skeletal muscle exacerbates sarcopenia and impairs autophagy by reducing AMPK signaling

Heme serves as a prosthetic group in hemoproteins, including subunits of the mammalian mitochondrial electron transfer chain. The first enzyme in vertebrate heme biosynthesis, 5-aminolevulinic acid synthase 1 (ALAS1), is ubiquitously expressed and essential for producing 5-aminolevulinic acid (ALA). We previously showed that Alas1 heterozygous mice at 20–35 weeks (aged-A1+/−s) manifested impaired glucose metabolism, mitochondrial malformation in skeletal muscle, and reduced exercise tolerance, potentially linked to autophagy dysfunction. In this study, we investigated autophagy in A1+/−s and a sarcopenic phenotype in A1+/−s at 75–95 weeks (senile-A1+/−s). Senile-A1+/−s exhibited significantly reduced body and gastrocnemius muscle weight, and muscle strength, indicating an accelerated sarcopenic phenotype. Decreases in total LC3 and LC3-II protein and Map1lc3a mRNA levels were observed in aged-A1+/−s under fasting conditions and in Alas1 knockdown myocyte-differentiated C2C12 cells (A1KD-C2C12s) cultured in high- or low-glucose medium. ALA treatment largely reversed these declines. Reduced AMP-activated protein kinase (AMPK) signaling was associated with decreased autophagy in aged-A1+/−s and A1KD-C2C12s. AMPK modulation using AICAR (activator) and dorsomorphin (inhibitor) affected LC3 protein levels in an AMPK-dependent manner. Our findings suggest that heme deficiency contributes to accelerated sarcopenia-like defects and reduced autophagy in skeletal muscle, primarily due to decreased AMPK signaling.

Association of Gene Polymorphisms and Serum Levels of ALAS1 with the Risk of Anti‐Tuberculosis Drug‐Induced Liver Injury

AbstractThe accumulation of protoporphyrin IX in the liver caused by isoniazid and rifampicin through the disorder of heme biosynthesis was considered an important mechanism of anti‐tuberculosis drug‐induced liver injury (ATLI). Alanine synthase 1 (ALAS1) is a rate‐limiting enzyme in the process of heme synthesis. This study aimed to investigate the association between ALAS1 gene polymorphism, serum ALAS1 level, and the risk of ATLI. This study was a case‐control study including 58 ATLI cases and 192 controls. Four single nucleotide polymorphisms (SNPs) of the ALAS1 gene were selected for genotyping and serum ALAS1 concentrations were detected using ELISA kits. There was no significant difference in the genotype distribution of four SNPs between the ATLI cases and the controls under different genetic models. Patients carrying the GG genotype of SNP rs352163 in controls had higher baseline ALAS1 levels than those in ATLI cases (243.6 vs 290.2 ng/L, P = .034), and patients with baseline ALAS1 < 337.85 ng/L had a higher risk of ATLI than those with ALAS1 ≥ 337.85 ng/L (HR = 2.679, 95% CI: 1.360‐5.278, P = .004). Our findings indicated that the serum ALAS1 concentrations in the ATLI cases were lower than those in the controls, and the lower baseline ALAS1 levels can be associated with higher ATLI risk.

The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis

BackgroundLoss-of-function mutations of ZBTB24 cause immunodeficiency, centromeric instability, and facial anomalies syndrome 2 (ICF2). ICF2 is a rare autosomal recessive disorder with immunological defects in serum antibodies and circulating memory B cells, resulting in recurrent and sometimes fatal respiratory and gastrointestinal infections. The genotype–phenotype correlation in patients with ICF2 indicates an essential role of ZBTB24 in the terminal differentiation of B cells.MethodsWe used the clustered regularly interspaced short palindromic repeats (CRISPER)/Cas9 technology to generate B cell specific Zbtb24-deficient mice and verified the deletion specificity and efficiency by quantitative polymerase chain reaction (Q-PCR) and western blotting analyses in fluorescence-activated cell sorting (FACS)-sorted cells. The development, phenotype of B cells and in vivo responses to T cell dependent or independent antigens post immunization were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Adoptive transfer experiment in combination with in vitro cultures of FACS-purified B cells and RNA-Seq analysis were utilized to specifically determine the impact of Zbtb24 on B cell biology as well as the underlying mechanisms.ResultsZbtb24 is dispensable for B cell development and maintenance in naive mice. Surprisingly, B cell specific deletion of Zbtb24 does not evidently compromise germinal center reactions and the resulting primary and secondary antibody responses induced by T cell dependent antigens (TD-Ags), but significantly inhibits T cell independent antigen-elicited antibody productions in vivo. At the cellular level, Zbtb24-deficiency specifically impedes the plasma cell differentiation of B1 cells without impairing their survival, activation and proliferation in vitro. Mechanistically, Zbtb24-ablation attenuates heme biosynthesis partially through mTORC1 in B1 cells, and addition of exogenous hemin abrogates the differentiation defects of Zbtb24-null B1 cells.ConclusionsZbtb24 seems to regulate antibody responses against TD-Ags B cell extrinsically, but it specifically promotes the plasma cell differentiation of B1 cells via heme synthesis in mice. Our study also suggests that defected B1 functions contribute to recurrent infections in patients with ICF2.

Metabolic Engineering of Escherichia coli for Production of a Bioactive Metabolite of Bilirubin.

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC-MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host.

Physiological Metabolic Analysis of VB12 Accumulation in Ensifer adhaerens Casida A Enhanced by Oxygen Limitation.

Cobalamin (VB12) is in enormous demand across the fields of medicine, food, and feed additives. However, the oxygen supply plays a critical role in VB12 biosynthesis by Ensifer adhaerens Casida A and has been identified as a bottleneck for economical substrate consumption. This study elucidates the relationship between oxygen limitation and VB12 accumulation with transcriptomic and metabolomic analyses. Under oxygen limitation, E. adhaerens enhances oxygen transport and storage by increasing expression of flavin hemoglobin (Hmp), which was up-regulated 6-fold at 24h of oxygen restriction compared to the oxygen restriction of 4h (p<0.01). Because of the cofactor of Hmp is heme, the demand for heme increases, leading to the upregulation of genes in the heme biosynthesis pathway. Similarly, genes involved in biosynthesis of its precursor, 5-ALA, were upregulated as well. 5-ALA is also a direct precursor of VB12, further leading to the upregulation of genes in the VB12 biosynthesis pathway. This process initiates biosynthesis and accumulation of VB12. As VB12 and heme biosynthesis progresses, genes associated with the biosynthesis and transportation pathways of compounds related to their biosynthesis were likewise upregulated, including genes involved in S-adenosyl methionine (SAM) biosynthesis, and the transport of Fe2+ and Co2+. Additionally, amino acids and organic acids associated with biosynthesis were also extensively consumed, such as methionine, which is used for synthesizing SAM, decreased by 310% after 24 h of oxygen limitation compared to 20% dissolved oxygen (p<0.05). At the same time, genes related to growth-associated metabolic pathways, such as pentose phosphate pathway (PPP), were significantly downregulated. Therefore, the potential mechanism by which E. adhaerens accumulates VB12 under oxygen-limited conditions by enhancing Hmp expression, which facilitates the porphyrin metabolic pathway and promotes VB12 biosynthesis. This research provides valuable insights for increasing VB12 production through metabolic engineering and process optimization.

Role of Mitochondrial Iron Uptake in Acetaminophen Hepatotoxicity.

Overdose of acetaminophen (APAP) produces fulminant hepatic necrosis. The underlying mechanism of APAP hepatotoxicity involves mitochondrial dysfunction, including mitochondrial oxidant stress and the onset of mitochondrial permeability transition (MPT). Reactive oxygen species (ROS) play an important role in APAP-induced hepatotoxicity, and iron is a critical catalyst for ROS formation. This review summarizes the role of mitochondrial ROS formation in APAP hepatotoxicity and further focuses on the role of iron. Normally, hepatocytes take up Fe3+-transferrin bound to transferrin receptors via endocytosis. Concentrated into lysosomes, the controlled release of iron is required for the mitochondrial biosynthesis of heme and non-heme iron-sulfur clusters. After APAP overdose, the toxic metabolite, NAPQI, damages lysosomes, causing excess iron release and the mitochondrial uptake of Fe2+ by the mitochondrial calcium uniporter (MCU). NAPQI also inhibits mitochondrial respiration to promote ROS formation, including H2O2, with which Fe2+ reacts to form highly reactive •OH through the Fenton reaction. •OH, in turn, causes lipid peroxidation, the formation of toxic aldehydes, induction of the MPT, and ultimately, cell death. Fe2+ also facilitates protein nitration. Targeting pathways of mitochondrial iron movement and consequent iron-dependent mitochondrial ROS formation is a promising strategy to intervene against APAP hepatotoxicity in a clinical setting.

PpIX-enabled fluorescence-based detection and photodynamic priming of platinum-resistant ovarian cancer cells under fluid shear stress.

Over 75% percent of ovarian cancer patients are diagnosed with advanced-stage disease characterized by unresectable intraperitoneal dissemination and the presence of ascites, or excessive fluid build-up within the abdomen. Conventional treatments include cytoreductive surgery followed by multi-line platinum and taxane chemotherapy regimens. Despite an initial response to treatment, over 75% of patients with advanced-stage ovarian cancer will relapse and succumb to platinum-resistant disease. Recent evidence suggests that fluid shear stress (FSS), which results from the movement of fluid such as ascites, induces epithelial-to-mesenchymal transition and confers resistance to carboplatin in ovarian cancer cells. This study demonstrates, for the first time, that FSS-induced platinum resistance correlates with increased cellular protoporphyrin IX (PpIX), the penultimate downstream product of heme biosynthesis, the production of which can be enhanced using the clinically approved pro-drug aminolevulinic acid (ALA). These data suggest that, with further investigation, PpIX could serve as a fluorescence-based biomarker of FSS-induced platinum resistance. Additionally, this study investigates the efficacy of PpIX-enabled photodynamic therapy (PDT) and the secretion of extracellular vesicles under static and FSS conditions in Caov-3 and NIH:OVCAR-3 cells, two representative cell lines for high-grade serous ovarian carcinoma (HGSOC), the most lethal form of the disease. FSS induces resistance to ALA-PpIX-mediated PDT, along with a significant increase in the number of EVs. Finally, the ability of PpIX-mediated photodynamic priming (PDP) to enhance carboplatin efficacy under FSS conditions is quantified. These preliminary findings in monolayer cultures necessitate additional studies to determine the feasibility of PpIX as a fluorescence-based indicator, and mediator of PDP, to target chemoresistance in the context of FSS.

Acute intermittent porphyria: a disease with low penetrance and high heterogeneity.

Acute intermittent porphyria (AIP) is caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS), a key enzyme in the heme biosynthesis pathway. AIP is an autosomal dominant disorder characterized by low penetrance and a highly heterogenous clinical presentation. The estimated prevalence of AIP is 5-10 cases per 100,000 persons, with acute attacks manifesting in less than 1% of the at-risk population. This low frequency of attacks suggests significant roles for oligogenic inheritance and environmental factors in the pathogenesis of the disease. In recent years, identification of several modifier genes has advanced our understanding of the factors influencing AIP penetrance and disease severity. This review summarizes these factors including the impact of specific HMBS mutations, oligogenic inheritance, mitochondrial DNA copy number, age, sex, the influence of sex hormones, and the role of environmental factors. Further studies into the etiology of AIP disease penetrance should inform pathogenesis, potentially allowing for the development of more precise diagnostic and therapeutic approaches.

Harnessing Porphyrin Accumulation in Liver Cancer: Combining Genomic Data and Drug Targeting.

The liver, a pivotal organ in human metabolism, serves as a primary site for heme biosynthesis, alongside bone marrow. Maintaining precise control over heme production is paramount in healthy livers to meet high metabolic demands while averting potential toxicity from intermediate metabolites, notably protoporphyrin IX. Intriguingly, our recent research uncovers a disrupted heme biosynthesis process termed 'porphyrin overdrive' in cancers that fosters the accumulation of heme intermediates, potentially bolstering tumor survival. Here, we investigate heme and porphyrin metabolism in both healthy and oncogenic human livers, utilizing primary human liver transcriptomics and single-cell RNA sequencing (scRNAseq). Our investigations unveil robust gene expression patterns in heme biosynthesis in healthy livers, supporting electron transport chain (ETC) and cytochrome P450 function without intermediate accumulation. Conversely, liver cancers exhibit rewired heme biosynthesis and a massive downregulation of cytochrome P450 gene expression. Notably, despite diminished drug metabolism, gene expression analysis shows that heme supply to the ETC remains largely unaltered or even elevated with patient cancer progression, suggesting a metabolic priority shift. Liver cancers selectively accumulate intermediates, which are absent in normal tissues, implicating their role in disease advancement as inferred by expression analysis. Furthermore, our findings in genomics establish a link between the aberrant gene expression of porphyrin metabolism and inferior overall survival in aggressive cancers, indicating potential targets for clinical therapy development. We provide in vitro proof-of-concept data on targeting porphyrin overdrive with a drug synergy strategy.