Hc-hrg-2, a glutathione transferase gene, regulates heme homeostasis in the blood-feeding parasitic nematode Haemonchus contortus

Zhou, Jingru; Bu, Dan-Ru; Zhao, Xianfeng; Wu, Fei; Chen, Xue-Qiu; Shi, Huiping; Yao, Chaoqun; Du, Aifang; Yang, Yi

doi:10.1186/s13071-020-3911-z

Cited by 10 publications

(7 citation statements)

References 44 publications

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“…Membranes were subsequently washed three times for 30 min with TBST and revealed with a solution containing 0.1 mg/mL 3,3 diaminobenzidine (DAB), 0.1% H 2 O 2 , 10 mM HEPES, pH 6.2, and 100 µM CaCl 2 overnight at 4 • C in the dark [66]. Alternatively, heme-binding proteins were evaluated by 1 h room temperature incubation of the protein extracts (20 µg) with hemin (300 µM) in 250 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 10% glycerol, followed by SDS-PAGE [67]. Then, gels were washed for 1 h with PBS containing Triton X-100 (2.5%) and equilibrated for 30 min with sodium acetate 0.5 M (pH 5.0).…”

Section: Sds-page and Hemin-binding Blotsmentioning

confidence: 99%

“…The chemiluminescent substrate Super Signal R West Pico was applied and immediately detected the signals generating a digital image file. The signal intensities were quantified using TotalLab (v 2009, Nonlinear Dynamics, Newcastle-Upon-Tyne, UK) software [67]. Binding motifs were considered analyzing the spots with signal intensity greater than or equal to 50% of the highest signal.…”

Section: Spot-synthesismentioning

confidence: 99%

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SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites

Lechuga

Souza-Silva

Sacramento

et al. 2021

IJMS

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(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC–MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC–MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus’s adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

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Section: Sds-page and Hemin-binding Blotsmentioning

confidence: 99%

Section: Spot-synthesismentioning

confidence: 99%

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites

Lechuga

Souza-Silva

Sacramento

et al. 2021

IJMS

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“…Oligonucleotide primers used for PCR-based molecular cloning are shown in S2 Table . Recombinant Hc-HRG-1 protein was purifed using a Ni-NTA agarose column (Qiagen, Beijing, China), and protein amount determined using the Bradford Protein Quantitation Kit (Fude Biological technology, Hangzhou, China). The haem binding assay was performed as described previously [15]. In brief, haemin chloride (10 μM) was mixed with Hc-HRG-1 (10 μM) and incubated at room temperature, shaking for 2 h. Haem binding was measured from three technical replicates by absorption in a Synergy HT Multimode Reader (BioTek, USA), and the mean value calculated.…”

Section: Haem Binding Assaymentioning

confidence: 99%

“…Apart from the identification of an HRG-1 orthologue in the parasitic nematode Brugia malayi-the causative agent of lymphatic filariasis of humans [12]-almost nothing is known about haem acquisition (i.e. transport into, within and between cells) in haematophagous and non-haematophagous parasitic nematodes [13][14][15]. Given that haem is essential for life, understanding the molecular and cellular biology of haem transporter(s) in these pathogens could lead to discovering ways of disrupting transporter function(s) for the purpose of blocking parasite development [5,7,13].…”

Section: Introductionmentioning

confidence: 99%

Haem transporter HRG-1 is essential in the barber’s pole worm and an intervention target candidate

Yang

Zhou

et al. 2023

PLoS Pathog

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Parasitic roundworms (nematodes) have lost genes involved in the de novo biosynthesis of haem, but have evolved the capacity to acquire and utilise exogenous haem from host animals. However, very little is known about the processes or mechanisms underlying haem acquisition and utilisation in parasites. Here, we reveal that HRG-1 is a conserved and unique haem transporter in a broad range of parasitic nematodes of socioeconomic importance, which enables haem uptake via intestinal cells, facilitates cellular haem utilisation through the endo-lysosomal system, and exhibits a conspicuous distribution at the basal laminae covering the alimentary tract, muscles and gonads. The broader tissue expression pattern of HRG-1 in Haemonchus contortus (barber’s pole worm) compared with its orthologues in the free-living nematode Caenorhabditis elegans indicates critical involvement of this unique haem transporter in haem homeostasis in tissues and organs of the parasitic nematode. RNAi-mediated gene knockdown of hrg-1 resulted in sick and lethal phenotypes of infective larvae of H. contortus, which could only be rescued by supplementation of exogenous haem in the early developmental stage. Notably, the RNAi-treated infective larvae could not establish infection or survive in the mammalian host, suggesting an indispensable role of this haem transporter in the survival of this parasite. This study provides new insights into the haem biology of a parasitic nematode, demonstrates that haem acquisition by HRG-1 is essential for H. contortus survival and infection, and suggests that HRG-1 could be an intervention target candidate in a range of parasitic nematodes.

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“…Membranes were subsequently washed three times for 30 min with TBST and revealed with a solution containing 0.1 mg/mL 3,3' diaminobenzidine (DAB), 0.1 % H2O2, 10 mM HEPES, pH 6.2, and 100 µM CaCl2 overnight at 4 °C in the dark [62]. Alternatively, hemebinding proteins were evaluated by 1 h room temperature incubation of the protein extracts (20 µg) with hemin (300 µM) in 250 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 10% glycerol, followed by SDS-PAGE [63]. Then, gels were washed for 1 h with PBS containing Triton X-100 (2.5 %) and equilibrated for 30 min with sodium acetate 0.5 M (pH 5.0).…”

Section: Sds-page and Hemin-binding Blotsmentioning

confidence: 99%

SARS-CoV-2 proteins bind heme and hemoglobin

De-Simone

Lechuga

Souza-Silva

et al. 2021

Preprint

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The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins’ possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemoglobin’s porphyrin coordination region. Different hemin binding capacities of mock and SARS-CoV-2-infected culture extracts were noticed using gel electrophoresis and TMB staining. Hemin-binding proteins were isolated from SARS-CoV-2-infected cells by affinity chromatography and identified by shotgun proteomics, indicating that structural (nucleoprotein, spike, and membrane protein) and non-structural (Nsp3 and Nsp7) viral proteins interact with hemin. In vitro analyses of virus adsorption to host cells and viral replication studies in Vero cells demonstrated inhibitory activities - at different levels - by hemin, protoporphyrin IX (PpIX) Hb. Strikingly, free Hb at 1mM suppressed viral replication (99 %), and its interaction with SARS-CoV-2 was localized to the RBD region of the Spike protein. The findings showed clear evidence of new avenues to disrupt viral replication and understand virus physiopathology that warrants further investigation.

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Hc-hrg-2, a glutathione transferase gene, regulates heme homeostasis in the blood-feeding parasitic nematode Haemonchus contortus

Cited by 10 publications

References 44 publications

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites

Haem transporter HRG-1 is essential in the barber’s pole worm and an intervention target candidate

SARS-CoV-2 proteins bind heme and hemoglobin

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