Coproporphyrinogen Oxidase Research Articles

Proteomic and phosphoproteomic analysis of a Haematococcus pluvialis (Chlorophyceae) mutant with a higher heterotrophic cell division rate reveals altered pathways involved in cell proliferation and nutrient partitioning.

Haematococcus pluvialis has been used to produce the ketocarotenoid antioxidant, astaxanthin. Currently, heterotrophic cultivation of H. pluvialis is limited by slow growth rates. This work aimed to address this challenge by exploring the mechanisms of acetate metabolism in Haematococcus. Chemical mutagenesis and screening identified H. pluvialis strain KREMS 23D-3 that achieved up to a 34.9% higher cell density than the wild type when grown heterotrophically on acetate. An integrative proteomics and phosphoproteomics approach was employed to quantify 4955 proteins and 5099 phosphorylation sites from 2505 phosphoproteins in the wild-type and mutant strains of H. pluvialis. Among them, 12 proteins were significantly upregulated and 22 significantly downregulated in the mutant while phosphoproteomic analysis identified 143 significantly upregulated phosphorylation sites on 106 proteins and 130 downregulated phosphorylation sites on 114 proteins. Upregulation of anaphase-promoting complex phosphoproteins and downregulation of a putative cell cycle division 20 phosphoprotein in the mutant suggests rapid mitotic progression, coinciding with higher cell division rates. Upregulated coproporphyrinogen oxidase and phosphorylated magnesium chelatase in the mutant demonstrated altered nitrogen partitioning toward chlorophyll biosynthesis. The large proportion of differentially expressed phosphoproteins suggests phosphorylation is a key regulator for protein expression and activity in Haematococcus. Taken together, this study reveals the regulation of interrelated acetate metabolic pathways in H. pluvialis and provides protein targets that may guide future strain engineering work.

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

Features of immunological indicators in children with allergy modified by nickel contamination of biomedia and polymorphism of CPOX rs1131857 detoxification gene

Introduction. The impact of unfavourable environmental chemical factors on the human immune system predetermines the conditions for the formation of pathological conditions associated with allergies.
 The purpose of the study. To study the features of immunological indicator parameters in children with allergies modified by contamination of biological media with nickel and polymorphism of the detoxification CPOX rs1131857 gene.
 Materials and methods. A study was conducted one hundred eighty children suffering from various forms of allergies, permanently residing in the zone of the Far North (121 of them — under conditions of aerogenic exposure to nickel aerosols, and 59 children — in a “conditionally clean” territory). The subject of the study was biological media (blood). The change in the content of specific IgE to nickel was determined by an allergosorbent test with an enzyme label. The study of cell differentiation clusters included the use of flow cytometry (CD3+, CD19+, CD3+CD4+, CD3+CD8+, CD16+CD56+). Polymorphism of the CPOX (rs1131857) gene was studied by real-time polymerase chain reaction (PCR).
 Results. The analysis of the immune and genetic status of children with various manifestations of allergic pathology revealed cellular immunodeficiency (CD3+ CD4+, a decrease in the immunoregulatory index), the development of processes of hypersensitivity to chemical technogenic haptens (IgE to nickel), as well as an increased frequency of occurrence of heterozygous AC polymorphism of the second detoxification gene. phase CPOX rs1131857, verified by significant parameters of inheritance models (OR = 2.83; 95% CI: 1.14–7.01; 
 p < 0.05).
 Limitations. The present study is not exposed to the adult population, does not consume the population, and does not consume, except in the Far North.
 Conclusion. A feature of the course of allergic pathology in the children’s population of the Arctic, modified by contamination of biomedia with nickel and polymorphism of the detoxification CPOX rs1131857 gene, will be an increased frequency and duration of recurrence of allergopathology due to the addition of viral (decrease in cellular immunity) and hapten (nickel) components.

The Molecular Evolution, Structure, and Function of Coproporphyrinogen Oxidase and Protoporphyrinogen Oxidase in Prokaryotes.

Coproporphyrinogen oxidase (CgoX) and protoporphyrinogen oxidase (PgoX) catalyze the oxidation of the flexible cyclic tetrapyrrole of porphyrinogen compounds into fully conjugated, planar macrocyclic porphyrin compounds during heme biosynthesis. These enzymes are activated via different pathways. CgoX oxidizes coproporphyrinogen III to coproporphyrin III in the coproporphyrin-dependent pathway, whereas PgoX oxidizes protoporphyrinogen IX to protoporphyrin IX in the penultimate step of the protoporphyrin-dependent pathway. The phylogenetic analysis presented herein demonstrates a clear differentiation between the two enzyme classes, as evidenced by the clustering of sequences in distinct clades, and it shows that, at the origin of porphyrinogen-type oxidase evolution, PgoXs from cyanobacteria were found, which were noticeably separated from descendant PgoX representatives of Deltaproteobacteria and all later PgoX variants, leading to many eukaryotic clades. CgoX sequences originating from the monoderm Actinomycetota and Bacillota were well separated from the predecessor clades containing PgoX types and represent a peculiar type of gene speciation. The structural similarities and differences between these two oxidases are discussed based on their protein sequence alignment and a structural comparison.

SAT292 Adrenal Insufficiency Associated With Biallelic Mutations In Porphyria Genes

Abstract Disclosure: C. Smith: None. A. Jackson: None. A. Al-Salihi: None. A. Janecke: None. E. Steichen: None. D. Darby: None. L. Griffin: None. S. Banka: None. S. Elsayed: None. L. Chan: None. L. Metherell: None. Adrenal insufficiency (AI) is life-threatening and can present alone or in combination with other co-morbidities. Here we describe families with a novel association of AI with porphyria caused by biallelic mutations in protoporphyrinogen oxidase (PPOX) or coproporphyrinogen oxidase (CPOX). The porphyrias are a group of disorders caused by defects in one of eight enzymes within the haem biosynthetic pathway, divided into acute porphyrias, resulting in mainly neurovisceral symptoms, or cutaneous porphyrias, mainly affecting the skin. Acute porphyria attacks can be life-threatening, resulting in permanent disability or death. Variegate porphyria and hereditary coproporphyria are associated with autosomal dominant mutations in PPOX and CPOX respectively, with biallelic inheritance rarely reported and knockout mice that are embryonic lethal.We performed whole exome sequencing (WES), array Comparative Genomic Hybridization (aCGH) and whole genome sequencing (WGS) in 3 families with AI and variegate or hereditary porphyria. In kindred 1, with 4 affected individuals, WES revealed a homozygous mutation, p.Glu339Lys, in PPOX. aCGH and WGS revealed no Copy Number Variants (CNVs) and no other variants, in genes causing AI, common to all affected individuals. In families 2 and 3, homozygous mutations in CPOX p.(Pro367Ala) in one patient and p.(Ser28Ter) in two siblings were identified by WGS and WES respectively, with a clinical picture of AI and hereditary coproporphyria. Unlike other acute porphyria kindreds, the heterozygous parents were asymptomatic, manifesting neither porphyria nor AI, suggesting that the level of enzyme function is key for both phenotypes.Reduced PPOX/CPOX activity could cause AI through either; (i) a dearth of haem for steroidogenic CYP450 enzyme action, (ii) toxicity of intermediate porphyrins or (iii) increased oxidative stress. To investigate the mechanism, we first created H295R, human adrenocortical cells, with differing degrees of PPOX-knockdown (KD) by shRNA. Proliferation, measured by MTT assay or GFP accumulation, was lower in PPOX-KD cells at 72 hours and mitochondrial respiration was diminished, possibly due to toxicity of porphyrin precursors. CYP11A1 expression was unaltered whereas STAR and CYP17A1 were significantly lower in PPOX-KD cells. GSH/GSSG ratio, a marker of oxidative stress, was lower in KD cells suggesting increased ROS in these cells. Finally, and definitively, knockdown of at least 60% PPOX protein in H295R cells reduced cortisol output by 2.2-fold (p<0.01) at baseline and 1.9-fold (p<0.0001) in forskolin stimulated cells. Similar CPOX studies are underway.These studies show that cortisol is reduced in individuals with biallelic PPOX/CPOX mutations and, in conjunction with previous cases of AI in association with other genes in the pathway, suggests adrenal function should be monitored in individuals with porphyria. Presentation: Saturday, June 17, 2023

A-324 Incorporating the Screening for Congenital Cytomegalovirus into a Newborn Genetic Testing Performed on Saliva Swab: An Intersection between Human Genetics and Infectious Diseases

Abstract Background Newborn genetic testing is a powerful tool for improving the future healthcare of infants because of its potential to screening for a wide range of disorders with a single test. The assay can be performed in non-invasive sample such as saliva swabs. Hundreds of genes associated to disorders with significant morbidity and mortality (if undiagnosed) are analyzed. Despite the knowledge that infectious disorders such as congenital cytomegalovirus virus (cCMV) infection also has potential to cause serious illness to newborns (10–15% of apparently asymptomatic infants with cCMV may develop hearing loss) most of the newborn genetic testing does not test the saliva for cCMV. This validation aims to compare two laboratory-developed PCR-based CMV tests in saliva samples to add the cCMV screening into our newborn genetic testing. Methods Saliva swabs (OCR-100, DNA Genotek) from infants of our NGS-based newborn genetic testing were enrolled (n = 171). Parent signed informed consent agreeing with the used of the left-over specimens. Infants age ranged from 0 to 10 years. Saliva DNA was extracted by using QIAsymphony and DSP DNA mini kit (Qiagen). CMV was tested by two independent qPCR assays performed on LightCycler 480II using LightCycler Multiplex DNA Master (Roche). Each assays target two separated regions of the CMV genome (UL55 + UL123 and UL34 + UL80.5). A third assay targeting human CPOX gene was used as process control. The assays limits of detection were calculated by applying probit regression to the qualitative results returned from a serial dilution (1:10) of a positive pool (2 310 000 UI/mL) into a negative pool of samples. The accuracies for CMV detection were estimated by the total, positive and negative agreement between the compared sets of assays. The assay with lower limit of detection were chosen and its precision was evaluated by testing a subset of 26 saliva swabs (6 positives and 20 negatives) during 3 distinct days. For accuracy and precision experiments, Cq values >35 (∼750 UI/mL) was considered as not detected. Results The limits of detections were 40.7 UI/mL (95% CI:19.2–62.27 UI/mL) for UL55 + UL123 and 184 UI/mL (95% CI:64–304 UI/mL) for UL34 + UL80.5. CMV was detected in 8 and not detected in 163 saliva swabs by either UL55 + UL123 and UL34 + UL80.5 resulting in total, positive and negative agreements of 100% (95% CI:97–100%), 100% (95% CI:67–100%) and 100% (95% CI:97–100%), respectively. The median (min-max) Cq values for UL55 + UL123 were 30.75 (21.39–34.42) and for UL34 + UL80.5 were 30.81 (20.53–34.1). In the assay precision evaluation, UL55 + UL123 returned the expected result in 26/26, 26/26 and 25/26 samples on day 1, 2 and 3, respectively. The exception was a borderline positive sample that reach the Cq cut-off value (>35) on day 3 evaluation. Conclusion The UL55 + UL123 assay had lower limit of detection for CMV compared UL34 + UL80.5 and was chosen to be used in our cCMV screening. Both assays showed complete agreement when applied to saliva swabs samples. UL55 + UL123 presented acceptable precisions for positive and negative results. The cCMV screening by qPCR is now performed in parallel to NGS in our newborn genetic testing. cCMV test is only performed for newborns with less than 21 days of life, as recommend by specific guidelines.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors thatare directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified whichare mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Physiological and proteomic analyses reveal the important role of arbuscular mycorrhizal fungi on enhancing photosynthesis in wheat under cadmium stress

Arbuscular mycorrhizal fungi (AMF) are important in the phytoremediation of cadmium (Cd). Improving photosynthesis under Cd stress helps to increase crop yields. However, the molecular regulatory mechanisms of AMF on photosynthetic processes in wheat (Triticum aestivum) under Cd stress remain unclear. This study utilized physiological and proteomic analyses to reveal the key processes and related genes of AMF that regulate photosynthesis under Cd stress. The results showed that AMF promoted the accumulation of Cd in the roots of wheat but significantly reduced the content of Cd in the shoots and grains. The photosynthetic rates, stomatal conductance, transpiration rates, chlorophyll content, and accumulation of carbohydrates under Cd stress were increased by AMF symbiosis. Proteomic analysis showed that AMF significantly induced the expression of two enzymes involved in the chlorophyll biosynthetic pathway (coproporphyrinogen oxidase and Mg-protoporphyrin IX chelatase), improved the expression of two proteins related to CO2 assimilation (ribulose-1,5-bisphosphate carboxylase and malic enzyme), and increased the expression of S-adenosylmethionine synthase, which positively regulates abiotic stress. Therefore, AMF may regulate photosynthesis under Cd stress by promoting chlorophyll biosynthesis, carbon assimilation, and S-adenosylmethionine metabolism.

An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX.

Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non-conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro-environment of the isoalloxazine ring of FAD for the substrate-FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non-conserved residues can form a conserved element to maintain the function of CPO or PPO.

On the haem auxotrophy of the soft tick Ornithodoros moubata

Genomes of ticks display reductions, to various extents, in genetic coding for enzymes of the haem biosynthetic pathway. Here, we mined available transcriptomes of soft tick species and identified transcripts encoding only half of the enzymes involved in haem biosynthesis. Transcripts identified across most species examined were those coding for porphobilinogen synthase, coproporphyrinogen oxidase, protoporphyrinogen oxidase, and ferrochelatase. Genomic retention of porphobilinogen synthase seems to be soft tick-restricted as no such homologue has been identified in any hard tick species. Bioinformatic mining is thus strongly indicative of the lack of biochemical capacity for de novo haem biosynthesis, suggesting a requirement for dietary haem. In the hard tick Ixodes ricinus, depletion of dietary haem, i.e. serum feeding, leads to oviposition of haem-free eggs, with no apparent embryogenesis and larvae formation. In this work, we show that serum-fed Ornithodoros moubata females, unlike those of I. ricinus, laid haem-containing eggs similarly to blood-fed controls, but only by a small proportion of the serum-fed females. To enhance the effect of dietary haem depletion, O. moubata ticks were serum-fed consecutively as last nymphal instars and females. These females laid eggs with profoundly reduced haem deposits, confirming the host origin of the haem. These data confirm the ability of soft ticks to take up and allocate host haem to their eggs in order to drive reproduction of the ticks.

BALB.NCT-Cpoxnct is a unique mouse model of hereditary coproporphyria

In humans, mutations in the coproporphyrinogen oxidase (CPOX) gene can result in hereditary coproporphyria (HCP), characterized by high levels of coproporphyrin excretion in the urine and feces, as well as acute neurovisceral and chronic cutaneous manifestations. Appropriate animal models for comprehending the precise pathogenesis mechanism of HCP have not been reported that show similarities in terms of gene mutation, reduced CPOX activity, excess coproporphyrin accumulation, and clinical symptoms. As previously discovered, the BALB.NCT-Cpoxnct mouse carries a hypomorphic mutation in the Cpox gene. Due to the mutation, BALB.NCT-Cpoxnct had a drastic increase in coproporphyrin in the blood and liver persistently from a young age. In this study, we found that BALB.NCT-Cpoxnct mice manifested HCP symptoms. Similar to HCP patients, BALB.NCT-Cpoxnct excreted an excessive amount of coproporphyrin and porphyrin precursors in the urine and displayed neuromuscular symptoms, such as a lack of grip strength and impaired motor coordination. Male BALB.NCT-Cpoxnct had nonalcoholic steatohepatitis (NASH)-like liver pathology and sclerodermatous skin pathology. A portion of male mice had liver tumors as well, whereas female BALB.NCT-Cpoxnct lacked these hepatic and cutaneous pathologies. In addition, we discovered that BALB.NCT-Cpoxnct exhibited microcytic anemia. These results indicate that BALB.NCT-Cpoxnct mice serve as the suitable animal model to help gain insight into the pathogenesis and therapy of HCP.

P07.04.B Heme biosynthesis factors and 5-ALA induced fluorescence: analysis of mRNA and protein expression in fluorescing and non-fluorescing gliomas

Abstract Background The intraoperative visualization of adult-type diffuse gliomas with 5-aminolevulinic acid (5-ALA) induced fluorescence is widely used in the neurosurgical field. While visible 5-ALA induced fluorescence is found in the majority of high-grade gliomas, most low-grade gliomas lack visible fluorescence during surgery. Recently, the heme biosynthesis pathway was identified as crucial influencing factor for presence of visible fluorescence since it metabolizes 5-ALA to fluorescing Protoporphyrin IX (PpIX). However, the exact alterations within the heme biosynthesis pathway resulting in visible 5-ALA induced fluorescence in gliomas are still unclear. The aim of the present study was thus to compare the mRNA and protein expression of promising intramitochondrial heme biosynthesis enzymes/transporters in glioma tissue samples of different fluorescence behavior. Material and Methods A total of 19 strongly fluorescing and 21 non-fluorescing tissue samples from neurosurgical adult-type diffuse gliomas (WHO grades II-IV) were included in the current analysis. In these samples, we investigated the mRNA expression by quantitative real time PCR and protein expression using immunohistochemistry of the intramitochondrial heme biosynthesis enzymes Coproporphyrinogen Oxidase (CPOX), Protoporphyrinogen Oxidase (PPOX), Ferrochelatase (FECH) and the transporter ATP-binding Cassette Subfamily B Member 2 (ABCG2). Results Regarding mRNA expression analysis, we found a significantly decreased ABCG2 expression in fluorescing specimens compared to non-fluorescing samples (p=0.001), whereas no difference in CPOX, PPOX and FECH was present. With respect to protein expression, significantly higher levels of CPOX (p=0.005), PPOX (p<0.01) and FECH (p=0.003) were detected in fluorescing samples. Similar to mRNA expression analysis, the protein expression of ABCG2 (p=0.001) was significantly lower in fluorescing samples. Conclusion Distinct alterations of the analyzed heme biosynthesis factors were found primarily on protein level. Our data indicate that heme biosynthesis pathway activity in general is enhanced in fluorescing gliomas with upregulation of PpIX generating enzymes and decreased ABCG2 mediated PpIX efflux outweighing the also increased further metabolization of PpIX to heme. Intramitochondrial heme biosynthesis factors thus constitute promising pharmacological targets to optimize intraoperative 5-ALA fluorescence visualization of usually non-fluorescing tumors such as low-grade gliomas.

Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid

5-ALA-mediated photodynamic therapy (PDT) has been developed around the heme biosynthesis physiological pathway. It is based on the external supplementation of 5 aminolevulinic acid (5-ALA), increasing the activity of the heme pathway and leading to a significant protoporphyrin IX (PpIX) accumulation. Interestingly, this metbolite accumulation is predominant in cancer cells, induced by a highly active metabolism, therefore limiting off-target side effects and increasing therapy specificity. Nevertheless, the intrinsic mechanism responsible of PpIX accumulation on cells following PDT is still unknown, limiting clinical therapy translation. In order to further understand the mechanisms behind 5-ALA-induced PDT, in this study we aimed to evaluate the proteome changes reported on the physiological heme pathway, in response to an external 5-ALA supplementation. We studied two different scenarios following 5-ALA treatment, 5-ALA accumulation (5-ALA metabolization into the heme pathway blocked with inhibitors) and accumulation of PpIX (normal heme pathway with 5-ALA supplementation). Therefore, we were able to characterize enzymatic changes and to describe bottlenecks in the pathway. Following mass spectrometry analysis, we reported significant differences between 5-ALA and PpIX effects on heme biosynthesis and regulation of degradation. 5-ALA accumulation significantly decreased porphobilinogen deaminase (HMBS) expression, while phorphyrins accumulation (PpIX) upregulated heme synthesis, specifically HMBS and uroporphyrinogen decarboxylase (UROD), and enhanced the enzymatic level of the heme degradation pathway, including Heme oxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA). Interestingly, porphyrins induced a significant downregulation effect on oxygen-dependent coproporphyrinogen-III oxidase (CPOX). In conclusion, in this study we demonstrated that porphyrins play the most relevant role in heme biosynthesis modulation, while 5-ALA alone (PDT substrate) is not responsible of the main changes observed in this pathway during PDT treatment. Understanding heme enzyme modulation would help to design a more rational approach for patient treatment in the clinic. AIM: Effect of 5-ALA and porphyrins on the different Heme biosynthesis and degradation enzymes.

Comparison of Production and Fluorescence Characteristics of Phycoerythrin from Three Strains of Porphyridium.

Phycoerythrin, a special photosynthetic pigment, is widely used as fluorescent dye and has lots of underlying beneficial effects on health. A marine red microalga Porphyridium is considered as the potential feedstock for phycoerythrin production. However, the phycoerythrin-related properties of Porphyridium have not been systematically evaluated, especially between the species of P. cruentum and P. purpureum. The present study aimed to evaluate the production and fluorescence characteristics of phycoerythrin of three strains of Porphyridium. The results showed that P. purpureum SCS-02 presented the highest biomass, phycoerythrin content and yield were 6.43 g L−1, 9.18% DW and 0.288 g L−1, respectively. There was no significant difference between P. purpureum and P. cruentum in α and β subunits amino acid sequences of phycoerythrin and in fluorescence characteristics. The high gene expression level of the key enzymes in phycoerythrobilin synthesis (porphobilinogen synthase and oxygen-dependent coproporphyrinogen-III oxidase) could be related to the high phycoerythrin content of Porphyridium. Based on systematic evaluation, P. purpureum SCS-02 was selected due to its high biomass and phycoerythrin yield. P. purpureum and P. cruentum were highly similar in the phylogenetic tree, as well as in fluorescence characteristics; therefore, it was speculated that they might be the same Porphyridium species.

Systemic Lupus Erythematosus and Hereditary Coproporphyria: Two Different Entities Diagnosed by WES in the Same Patient

Background Hereditary coproporphyria (HCP) is a rare autosomal dominant disorder caused by a partial deficiency of coproporphyrinogen III oxidase (CPOX), and systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic predisposition. SLC7A7 (solute carrier family 7 member 7) may be associated with monogenic lupus disease; however, only 2 cases of concomitant HCP and SLE have been reported. Methods We report a 30-year-old woman with a six-year history of SLE presenting with abdominal pain, vomiting, dysuria, tachycardia, and hyponatremia. Whole exome sequencing (WES) and Sanger sequencing were carried out for the proband and members of her pedigree to detect the genetic background. The Gene Expression Omnibus (GEO) database was used to search the related gene expression profiles. Differentially expressed genes (DEGs) were identified using GEO2R. Result A novel heterozygous splicing mutation of CPOX (NM_000097): c.700+2 T > C (intron 2) was detected by WES in the proband, and it was considered likely pathogenic (PSV1+PM2). Sanger sequencing verified the heterozygous mutation of CPOX in the proband, although it was not detected in her father. WES also identified 62 other gene variants, especially two heterozygous variants in SLC7A7 (NM_001126106): c.250G > A (p. V84I) and c.625+1G > A (splicing). DEGs were detected from GSE51997, and the expression of CPOX was downregulated in SLE patients compared with normal controls (adj. P = 0.0071, logFC = −1.0975). Conclusion This study presents the first reported case of SLE coexisting with HCP in China; moreover, a novel splicing mutation of CPOX, i.e., c.700+2 T > C (intron 2), and two heterozygous mutations of SLC7A7 were reported. The simultaneous mutations of CPOX and SLC7A7 may explain the etiopathogenetic connections of HCP and SLE.

Heme Biosynthesis Factors and 5-ALA Induced Fluorescence: Analysis of mRNA and Protein Expression in Fluorescing and Non-fluorescing Gliomas.

ObjectiveThe intraoperative visualization of adult-type diffuse gliomas with 5-aminolevulinic acid (5-ALA) induced fluorescence is widely used in the neurosurgical field. While visible 5-ALA induced fluorescence is found in the majority of high-grade gliomas, most low-grade gliomas lack visible fluorescence during surgery. Recently, the heme biosynthesis pathway was identified as crucial influencing factor for presence of visible fluorescence since it metabolizes 5-ALA to fluorescing Protoporphyrin IX (PpIX). However, the exact alterations within the heme biosynthesis pathway resulting in visible 5-ALA induced fluorescence in gliomas are still unclear. The aim of the present study was thus to compare the mRNA and protein expression of promising intramitochondrial heme biosynthesis enzymes/transporters in glioma tissue samples of different fluorescence behavior.MethodsA total of 19 strongly fluorescing and 21 non-fluorescing tissue samples from neurosurgical adult-type diffuse gliomas (WHO grades II-IV) were included in the current analysis. In these samples, we investigated the mRNA expression by quantitative real time PCR and protein expression using immunohistochemistry of the intramitochondrial heme biosynthesis enzymes Coproporphyrinogen Oxidase (CPOX), Protoporphyrinogen Oxidase (PPOX), Ferrochelatase (FECH), and the transporter ATP-binding Cassette Subfamily B Member 2 (ABCG2).ResultsRegarding mRNA expression analysis, we found a significantly decreased ABCG2 expression in fluorescing specimens compared to non-fluorescing samples (p = 0.001), whereas no difference in CPOX, PPOX and FECH was present. With respect to protein expression, significantly higher levels of CPOX (p = 0.005), PPOX (p < 0.01) and FECH (p = 0.003) were detected in fluorescing samples. Similar to mRNA expression analysis, the protein expression of ABCG2 (p = 0.001) was significantly lower in fluorescing samples.ConclusionDistinct alterations of the analyzed heme biosynthesis factors were found primarily on protein level. Our data indicate that heme biosynthesis pathway activity in general is enhanced in fluorescing gliomas with upregulation of PpIX generating enzymes and decreased ABCG2 mediated PpIX efflux outweighing the also increased further metabolization of PpIX to heme. Intramitochondrial heme biosynthesis factors thus constitute promising pharmacological targets to optimize intraoperative 5-ALA fluorescence visualization of usually non-fluorescing tumors such as low-grade gliomas.

Identification of a coproporphyrinogen-III oxidase gene and its correlation with nacre color in Hyriopsis cumingii.

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis.

Functional Diversity of HemN-like Proteins.

HemN is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the anaerobic oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, a key intermediate in heme biosynthesis. Proteins homologous to HemN (HemN-like proteins) are widespread in both prokaryotes and eukaryotes. Although these proteins are in most cases annotated as anaerobic coproporphyrinogen III oxidases (CPOs) in the public database, many of them are actually not CPOs but have diverse functions such as methyltransferases, cyclopropanases, heme chaperones, to name a few. This Perspective discusses the recent advances in the understanding of HemN-like proteins, and particular focus is placed on the diverse chemistries and functions of this growing protein family.

A novel ENU-induced Cpox mutation causes microcytic hypochromic anemia in mice.

Mouse models of red blood cell abnormalities are important for understanding the underlying molecular mechanisms of human erythrocytic diseases. DBA.B6-Mha (Microcytic hypochromic anemia) congenic mice were generated from the cross between N-ethyl-N-nitrosourea (ENU)-mutagenized male C57BL/6J and female DBA/2J mice as part of the RIKEN large-scale ENU mutagenesis project. The mice were established by backcrossing with DBA/2J mice for more than 20 generations. These mice showed autosomal-dominant microcytic hypochromic anemia with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels and increased red blood cell distribution width (RDW) and plasma ferritin levels. Linkage analysis indicated that the Mha locus was located within an interval of approximately 1.95-Mb between D16Nut1 (58.35 Mb) and D16Mit185 (60.30 Mb) on mouse chromosome 16. Mutation analysis revealed that DBA.B6-Mha mice had a point mutation (c.921-2A>G) at the acceptor site of intron 4 in the coproporphyrinogen oxidase (Cpox) gene, a heme-synthesizing gene. RT-PCR revealed that the Cpox mRNA in DBA.B6-Mha mice caused splicing errors. Our results suggest that microcytic hypochromic anemia in DBA.B6-Mha mice is owing to impaired heme synthesis caused by splice mutations in Cpox. Therefore, the DBA.B6-Mha mice may be used to elucidate the molecular mechanisms underlying microcytic hypochromic anemia caused by mutations in Cpox. Although low MCV levels are known to confer malarial resistance to the host, there were no marked changes in the susceptibility of DBA.B6-Mha mice to rodent malarial (Plasmodium yoelii 17XL) infection.

Involvement of increased endoplasmic reticulum stress in the development of cataracts in BALB.NCT-Cpoxnct mice

The BALB.NCT-Cpoxnct is a mutant mouse model for hereditary cataracts. We previously uncovered that the primary cause of the cataracts of BALB.NCT-Cpoxnct is a mutation in the coproporphyrinogen oxidase (Cpox) gene. Because of the mutation, excessive coproporphyrin is accumulated in the BALB.NCT-Cpoxnct lens. In this study, we analyzed the changes in transcriptome and proteins in the lenses of 4- and 12-week-old BALB.NCT-Cpoxnct to further elucidate the molecular etiology of cataracts in this mouse strain. Transcriptome analysis revealed that endoplasmic reticulum (ER) stress was increased in the BALB.NCT-Cpoxnct lens that induced persistent activation of the PERK signaling pathway of the ER stress response. Also, levels of crystallin transcripts and proteins were reduced in the BALB.NCT-Cpoxnct lens. Analysis of proteins disclosed aggregation of crystallins and keratins prior to the manifestation of cataracts in 4-week-old BALB.NCT-Cpoxnct mice. At 12 weeks of age, insoluble crystallins were accumulated in the cataractous BALB.NCT-Cpoxnct lens. Overall, our data suggest the following sequence of events in the BALB.NCT-Cpoxnct lens: accumulated coproporphyrin induces the aggregation of proteins including crystallins. Aggregated proteins increase ER stress that, in turn, leads to the repression of global translation of proteins including crystallins. The decline in the molecular chaperone crystallin aggravates aggregation and insolubilization of proteins. This vicious cycle would eventually lead to cataracts in BALB.NCT-Cpoxnct.