scholarly journals Haem-responsive gene transporter enables mobilization of host haem in ticks

Open Biology ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 210048
Author(s):  
J. Perner ◽  
T. Hatalova ◽  
M. Cabello-Donayre ◽  
V. Urbanova ◽  
D. Sojka ◽  
...  
Keyword(s):  
Protoporphyrin Ix ◽  
Protein Product ◽  
Yeast Growth ◽  
Free Living ◽  
Membrane Feeding ◽  
Haem Biosynthesis ◽  
Bona Fide ◽  
Solute Carrier ◽  
Responsive Gene ◽  
Systemic Rna

Ticks, notorious blood-feeders and disease-vectors, have lost a part of their genetic complement encoding haem biosynthetic enzymes and are, therefore, dependent on the acquisition and distribution of host haem. Solute carrier protein SLC48A1, aka haem-responsive gene 1 protein (HRG1), has been implicated in haem transport, regulating the availability of intracellular haem. HRG1 transporter has been identified in both free-living and parasitic organisms ranging from unicellular kinetoplastids, nematodes, up to vertebrates. However, an HRG1 homologue in the arthropod lineage has not yet been identified. We have identified a single HRG1 homologue in the midgut transcriptome of the tick Ixodes ricinus, denoted as Ir HRG, and have elucidated its role as a haem transporter. Data from haem biosynthesis-deficient yeast growth assays, systemic RNA interference and the evaluation of gallium protoporphyrin IX-mediated toxicity through tick membrane feeding clearly show that Ir HRG is the bona fide tetrapyrrole transporter. We argue that during evolution, ticks profited from retaining a functional hrg1 gene in the genome because its protein product facilitates host haem escort from intracellularly digested haemoglobin, rendering haem bioavailable for a haem-dependent network of enzymes.

Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)

2002 ◽  
Vol 362 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Johanna E. CORNAH ◽  
Jennifer M. ROPER ◽  
Davinder Pal SINGH ◽  
Alison G. SMITH
Keyword(s):  
Ferrous Iron ◽  
Specific Activity ◽  
Higher Plants ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Marker Enzyme ◽  
Hexane Extraction ◽  
Higher Plant ◽  
Major Site ◽  
Haem Biosynthesis

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.

scholarly journals A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

2020 ◽  
Vol 19 (12) ◽  
pp. 1968-1985
Author(s):  
Yi Liu ◽  
Michael J. Trnka ◽  
Shenheng Guan ◽  
Doyoung Kwon ◽  
Do-Hyung Kim ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Nuclear Import ◽  
Cell Model ◽  
Protoporphyrin Ix ◽  
Zinc Protoporphyrin ◽  
Primary Hepatocytes ◽  
Protein Aggregate ◽  
Nuclear Entry ◽  
Antibody Recognition ◽  
Bona Fide

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

scholarly journals Evolutionary origins of metabolic compartmentalization in eukaryotes

2010 ◽  
Vol 365 (1541) ◽  
pp. 847-855 ◽  
Author(s):  
William Martin
Keyword(s):  
Protein Targeting ◽  
Protein Product ◽  
Eukaryotic Cells ◽  
Evolutionary Time ◽  
Free Living ◽  
Evolutionary Origins ◽  
Dual Targeting ◽  
Targeting Signals ◽  
A Minor ◽  
Host Genes

Many genes in eukaryotes are acquisitions from the free-living antecedents of chloroplasts and mitochondria. But there is no evolutionary ‘homing device’ that automatically directs the protein product of a transferred gene back to the organelle of its provenance. Instead, the products of genes acquired from endosymbionts can explore all targeting possibilities within the cell. They often replace pre-existing host genes, or even whole pathways. But the transfer of an enzymatic pathway from one compartment to another poses severe problems: over evolutionary time, the enzymes of the pathway acquire their targeting signals for the new compartment individually, not in unison. Until the whole pathway is established in the new compartment, newly routed individual enzymes are useless, and their genes will be lost through mutation. Here it is suggested that pathways attain novel compartmentation variants via a ‘minor mistargeting’ mechanism. If protein targeting in eukaryotic cells possesses enough imperfection such that small amounts of entire pathways continuously enter novel compartments, selectable units of biochemical function would exist in new compartments, and the genes could become selected. Dual-targeting of proteins is indeed very common within eukaryotic cells, suggesting that targeting variation required for this minor mistargeting mechanism to operate exists in nature.

Terminal steps of haem biosynthesis

2002 ◽  
Vol 30 (4) ◽  
pp. 590-595 ◽  
Author(s):  
H. A. Dailey
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Ferrous Iron ◽  
Protoporphyrin Ix ◽  
Oxidative Decarboxylation ◽  
Protoporphyrinogen Oxidase ◽  
Coproporphyrinogen Oxidase ◽  
Cellular Location ◽  
Haem Biosynthesis ◽  
Small Set

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (co-proporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.

scholarly journals Dimeric Brucella abortus Irr protein controls its own expression and binds haem

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3427-3433 ◽  
Author(s):  
Marcela Martínez ◽  
Rodolfo A. Ugalde ◽  
Marta Almirón
Keyword(s):  
Brucella Abortus ◽  
Biosynthetic Pathway ◽  
Response Regulator ◽  
Protoporphyrin Ix ◽  
Close Relative ◽  
Iron Availability ◽  
Iron Deprivation ◽  
Haem Biosynthesis ◽  
Insight Into

Brucella abortus needs to synthesize haem in order to replicate intracellularly and to produce virulence in mice. Thus, to gain insight into the pathogenesis of the bacterium, regulatory proteins of the haem biosynthetic pathway were sought. An iron response regulator (Irr) from Bradyrhizobium japonicum, which is a close relative of Brucella, was previously described as being involved in the coordination of haem biosynthesis and iron availability. The Bru. abortus genome was searched for an irr orthologue gene, and the Bru. abortus irr gene was cloned, sequenced and disrupted. A null mutant was constructed that accumulated protoporphyrin IX under conditions of iron deprivation. This phenotype was overcome by a complementing plasmid carrying the wild-type irr. Purified recombinant Bru. abortus Irr behaved as a stable dimer and bound haem. Interestingly, in vivo, Irr was only detected in cells obtained from iron-limited cultures and the protein downregulated its own transcription. Through lacZ fusion, it was demonstrated that iron did not regulate irr transcription. The data reported show that Bru. abortus Irr is a homodimeric protein that is accumulated in iron-limited cells, controls its own transcription and downregulates the biosynthesis of haem precursors.

scholarly journals Inhibition of ferrochelatase during differentiation of murine erythroleukaemia cells

1987 ◽  
Vol 243 (2) ◽  
pp. 419-424 ◽  
Author(s):  
A Fadigan ◽  
H A Dailey
Keyword(s):  
Biosynthetic Pathway ◽  
Protoporphyrin Ix ◽  
Inhibitory Effect ◽  
Dimethyl Sulphoxide ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Bivalent Cations ◽  
Haem Biosynthesis ◽  
Low Levels ◽  
Rate Limiting

During dimethyl sulphoxide-induced differentiation of DS-19 murine erythroleukaemia (MEL) cells, the activity of the terminal enzyme of the haem-biosynthetic pathway, ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), is thought to be the rate-limiting step for haem production. Differentiation of induced MEL cells in the presence of exogeneously supplied protoporphyrin IX showed that total haem production was affected by added porphyrin only after 48 h. These data suggest that iron insertion, the terminal step, is rate-limiting during the first 48 h of differentiation. Addition of low levels of diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine to differentiating cultures resulted in decreased haem production and decreased ferrochelatase activity. N-Methylprotoporphyrin at nanomolar concentrations also strongly inhibited ferrochelatase activity, but had no inhibitory effect on cellular haem production. The bivalent cations Co2+, Cd2+ and Mn2+ were tested for their effect on haem production and ferrochelatase activity. All three metals were found to inhibit both haem formation and ferrochelatase activity, with Mn2+ being the strongest effector. These data, together with those previously published, suggest that the terminal step in haem biosynthesis is rate-limiting during the early stages of differentiation in MEL cells.

scholarly journals The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron-binding site

2017 ◽  
Vol 474 (20) ◽  
pp. 3513-3522 ◽  
Author(s):  
Charlie Hobbs ◽  
James D. Reid ◽  
Mark Shepherd
Keyword(s):  
Staphylococcus Aureus ◽  
Ferrous Iron ◽  
Substrate Inhibition ◽  
Iron Concentration ◽  
Protoporphyrin Ix ◽  
Site Directed Mutagenesis ◽  
Bacterial Phyla ◽  
Haem Biosynthesis ◽  
And Staphylococcus Aureus ◽  
Dependent Pathway

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.

scholarly journals Acquisition of exogenous haem is essential for tick reproduction

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jan Perner ◽  
Roman Sobotka ◽  
Radek Sima ◽  
Jitka Konvickova ◽  
Daniel Sojka ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Serum Proteins ◽  
Current Knowledge ◽  
Protein S ◽  
Gene Encoding ◽  
Membrane Feeding ◽  
Haem Biosynthesis ◽  
Tick Reproduction ◽  
Haem Oxygenase ◽  
Egg Fertility

Haem and iron homeostasis in most eukaryotic cells is based on a balanced flux between haem biosynthesis and haem oxygenase-mediated degradation. Unlike most eukaryotes, ticks possess an incomplete haem biosynthetic pathway and, together with other (non-haematophagous) mites, lack a gene encoding haem oxygenase. We demonstrated, by membrane feeding, that ticks do not acquire bioavailable iron from haemoglobin-derived haem. However, ticks require dietary haemoglobin as an exogenous source of haem since, feeding with haemoglobin-depleted serum led to aborted embryogenesis. Supplementation of serum with haemoglobin fully restored egg fertility. Surprisingly, haemoglobin could be completely substituted by serum proteins for the provision of amino-acids in vitellogenesis. Acquired haem is distributed by haemolymph carrier protein(s) and sequestered by vitellins in the developing oocytes. This work extends, substantially, current knowledge of haem auxotrophy in ticks and underscores the importance of haem and iron metabolism as rational targets for anti-tick interventions.

scholarly journals The HemQ coprohaem decarboxylase generates reactive oxygen species: implications for the evolution of classical haem biosynthesis

2016 ◽  
Vol 473 (21) ◽  
pp. 3997-4009 ◽  
Author(s):  
Charlie Hobbs ◽  
Harry A. Dailey ◽  
Mark Shepherd
Keyword(s):  
Reactive Oxygen Species ◽  
Biosynthetic Pathway ◽  
Protein Complexes ◽  
Protoporphyrin Ix ◽  
Reactive Oxygen ◽  
Divergent Evolution ◽  
Oxygen Species ◽  
Compound I ◽  
Fenton Chemistry ◽  
Haem Biosynthesis

Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes, including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates, including non-metallated porphyrins, such as protoporphyrin IX, which is well known to generate reactive oxygen species in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin. Here, we demonstrate, for the first time, that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyrin III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporphyrin IX (but not coproporphyrin III). Assays with hydrogen peroxide, horseradish peroxidase, superoxide dismutase, and catalase confirm that this stimulatory effect is mediated by superoxide. Structural modelling reveals that HemQ enzymes do not possess the structural attributes that are common to peroxidases that form compound I [FeIV==O]+, which taken together with the superoxide data leaves Fenton chemistry as a likely route for the superoxide-mediated stimulation of protoporphyrinogen IX oxidase activity of HemY. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. This work has implications for the divergent evolution of haem biosynthesis in ancestral microorganisms, and provides new structural and mechanistic insights into a recently discovered oxidative decarboxylase reaction.

Ultrastructure of neuromuscular relationships in the canine heartworm (dirofilaria immitis)

1986 ◽  
Vol 44 ◽  
pp. 278-279
Author(s):  
W. L. Steffens ◽  
Nancy B. Roberts ◽  
J. M. Bowen
Keyword(s):  
Dirofilaria Immitis ◽  
Domestic Dogs ◽  
Structural Description ◽  
Pharmacological Effects ◽  
Free Living ◽  
Canine Heartworm ◽  
The World ◽  
Synaptic Region ◽  
Muscle Innervation ◽  
Insight Into

The canine heartworm is a common and serious nematode parasite of domestic dogs in many parts of the world. Although nematode neuroanatomy is fairly well documented, the emphasis has been on sensory anatomy and primarily in free-living soil species and ascarids. Lee and Miller reported on the muscular anatomy in the heartworm, but provided little insight into the peripheral nervous system or myoneural relationships. The classical fine-structural description of nematode muscle innervation is Rosenbluth's earlier work in Ascaris. Since the pharmacological effects of some nematacides currently being developed are neuromuscular in nature, a better understanding of heartworm myoneural anatomy, particularly in reference to the synaptic region is warranted.

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