The essential role of the transporter ABCG2 in the pathophysiology of erythropoietic protoporphyria

Pengcheng Wang; Madhav Sachar; Jie Lu; Amina I. Shehu; Junjie Zhu; Jing Chen; Ke Liu; Karl E. Anderson; Wen Xie; Frank J. Gonzalez; Curtis D. Klaassen; Xiaochao Ma

doi:10.1126/sciadv.aaw6127

The essential role of the transporter ABCG2 in the pathophysiology of erythropoietic protoporphyria

Science Advances ◽

10.1126/sciadv.aaw6127 ◽

2019 ◽

Vol 5 (9) ◽

pp. eaaw6127 ◽

Cited By ~ 3

Author(s):

Pengcheng Wang ◽

Madhav Sachar ◽

Jie Lu ◽

Amina I. Shehu ◽

Junjie Zhu ◽

...

Keyword(s):

Essential Role ◽

Protoporphyrin Ix ◽

Heme Biosynthesis ◽

Biosynthesis Pathway ◽

Efflux Transporter ◽

Inherited Disease ◽

Loss Of Function ◽

Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited disease caused by loss-of-function mutations of ferrochelatase, an enzyme in the heme biosynthesis pathway that converts protoporphyrin IX (PPIX) into heme. PPIX accumulation in patients with EPP leads to phototoxicity and hepatotoxicity, and there is no cure. Here, we demonstrated that the PPIX efflux transporter ABCG2 (also called BCRP) determines EPP-associated phototoxicity and hepatotoxicity. We found that ABCG2 deficiency decreases PPIX distribution to the skin and therefore prevents EPP-associated phototoxicity. We also found that ABCG2 deficiency protects against EPP-associated hepatotoxicity by modulating PPIX distribution, metabolism, and excretion. In summary, our work has uncovered an essential role of ABCG2 in the pathophysiology of EPP, which suggests the potential for novel strategies in the development of therapy for EPP.

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Mutation in humanCLPXelevates levels ofδ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700632114 ◽

2017 ◽

Vol 114 (38) ◽

pp. E8045-E8052 ◽

Cited By ~ 26

Author(s):

Yvette Y. Yien ◽

Sarah Ducamp ◽

Lisa N. van der Vorm ◽

Julia R. Kardon ◽

Hana Manceau ◽

...

Keyword(s):

Protoporphyrin Ix ◽

Heme Biosynthesis ◽

Loss Of Function ◽

Dominant Mutation ◽

Erythropoietic Protoporphyria ◽

Aminolevulinate Synthase ◽

Abnormal Accumulation ◽

Reduced Activity ◽

Low Activity

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation inCLPXinactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

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Hox13 genes are required for mesoderm formation and axis elongation during early zebrafish development

Development ◽

10.1242/dev.185298 ◽

2020 ◽

Vol 147 (22) ◽

pp. dev185298

Author(s):

Zhi Ye ◽

David Kimelman

Keyword(s):

Retinoic Acid ◽

Essential Role ◽

Vertebrate Development ◽

Loss Of Function ◽

The People ◽

Vertebrate Embryo ◽

Mesoderm Formation ◽

Axis Elongation ◽

Null Mutants

ABSTRACTThe early vertebrate embryo extends from anterior to posterior due to the addition of neural and mesodermal cells from a neuromesodermal progenitor (NMp) population located at the most posterior end of the embryo. In order to produce mesoderm throughout this time, the NMps produce their own niche, which is high in Wnt and low in retinoic acid. Using a loss-of-function approach, we demonstrate here that the two most abundant Hox13 genes in zebrafish have a novel role in providing robustness to the NMp niche by working in concert with the niche-establishing factor Brachyury to allow mesoderm formation. Mutants lacking both hoxa13b and hoxd13a in combination with reduced Brachyury activity have synergistic posterior body defects, in the strongest case producing embryos with severe mesodermal defects that phenocopy brachyury null mutants. Our results provide a new way of understanding the essential role of the Hox13 genes in early vertebrate development.This article has an associated ‘The people behind the papers’ interview.

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Brain and Breast Cancer Cells with PTEN Loss of Function Demonstrate Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Using 3D Printed Scaffolds and Aligned Microfiber Tracts

10.21203/rs.3.rs-826338/v1 ◽

2021 ◽

Author(s):

Annalena Wieland ◽

Pamela L. Strissel ◽

Hannah Schorle ◽

Ezgi Bakirci ◽

Dieter Janzen ◽

...

Keyword(s):

Breast Cancer ◽

Cell Morphology ◽

Live Cell Imaging ◽

Essential Role ◽

Cell Imaging ◽

Loss Of Function ◽

Pten Loss ◽

3D Printed ◽

And Migration

Abstract Background: Glioblastoma multiforme (GBM) and triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with very poor patient outcomes. GBM uses axons and vessels as migratory cues to disseminate, however it is not known, if TNBC shares the same behavior. There is a need to understand brain tumor cell spreading and if GBM and TNBC have similar migration properties involving the signaling pathway RHOB-ROCK-PTEN. We tested for durotaxis, adherence and migration of GBM and TNBC using live-cell imaging and performed molecular analyses on three-dimensional (3D) structures.Methods: Aligned 3D printed scaffolds and microfibers were designed to mimic brain axon tracts and vessels for migration. GBM and TNBC cell lines, each with opposing PTEN genotypes, were analysed with RHO, ROCK and PTEN inhibitors and rescuing PTEN function using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while SEM, confocal microscopy and cell tracking addressed cell morphology. Results: GBM and TNBC with homozygote PTEN loss of function and RHOB high expression were amoeboid shaped and demonstrated enhanced durotaxis, adhesion and migration on 3D microfibers, in contrast to PTEN wildtype GBM and TNBC showing elongated cells and low RHOB. RNA-sequencing exhibited that RHOB was significantly the highest expressed gene in GBM PTEN loss of function cells. Pathway inhibitors and PTEN rescue of function verified an essential role of RHOB-ROCK-PTEN signaling for durotaxis, adhesion, migration, cell morphology and plasticity using 3D printed microfibers. Conclusions: This study validates a significant role of a PTEN genotype for cellular properties including durotaxis, adhesion and migration. GBM and TNBC cells with PTEN loss of function have a greater affinity for stiffer brain structures promoting metastasis. We propose the significance of PTEN and RHOB in cellular oncology not only for primary tumors, but also for metastasizing tumors, where RHOB inhibitors could play an essential role for improved therapy.

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The essential role of PRAK in tumor metastasis and its therapeutic potential

Nature Communications ◽

10.1038/s41467-021-21993-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yuqing Wang ◽

Wei Wang ◽

Haoming Wu ◽

Yu Zhou ◽

Xiaodan Qin ◽

...

Keyword(s):

Tumor Metastasis ◽

Essential Role ◽

Therapeutic Potential ◽

Lung Metastases ◽

Breast Cancers ◽

Loss Of Function ◽

Apparent Effect ◽

Metastatic Risk ◽

Pronounced Inhibition

AbstractMetastasis is the leading cause of cancer-related death. Despite the recent advancements in cancer treatment, there is currently no approved therapy for metastasis. The present study reveals a potent and selective activity of PRAK in the regulation of tumor metastasis. While showing no apparent effect on the growth of primary breast cancers or subcutaneously inoculated tumor lines, Prak deficiency abrogates lung metastases in PyMT mice or mice receiving intravenous injection of tumor cells. Consistently, PRAK expression is closely associated with metastatic risk in human cancers. Further analysis indicates that loss of function of PRAK leads to a pronounced inhibition of HIF-1α protein synthesis, possibly due to reduced mTORC1 activities. Notably, pharmacological inactivation of PRAK with a clinically relevant inhibitor recapitulates the anti-metastatic effect of Prak depletion, highlighting the therapeutic potential of targeting PRAK in the control of metastasis.

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Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011605 ◽

2019 ◽

Vol 295 (6) ◽

pp. 1539-1550 ◽

Cited By ~ 5

Author(s):

Edwin T. Tjhin ◽

Jenni A. Hayward ◽

Geoffrey I. McFadden ◽

Giel G. van Dooren

Keyword(s):

Oxygen Consumption ◽

Toxoplasma Gondii ◽

Heme Biosynthesis ◽

Covalent Attachment ◽

Biosynthesis Pathway ◽

Extracellular Acidification ◽

Mitochondrial Oxygen Consumption ◽

Atp Levels ◽

Free Heme

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.

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Role of the antioxidant system in the regulation of the chlorophyll biosynthesis pathway in the vascular plant Cucumis sativus

Functional Plant Biology ◽

10.1071/fp16393 ◽

2018 ◽

Vol 45 (4) ◽

pp. 464

Author(s):

Aarti Dhepe ◽

Komal Joshi

Keyword(s):

Cucumis Sativus ◽

Antioxidant System ◽

Chlorophyll Biosynthesis ◽

Vascular Plant ◽

Protoporphyrin Ix ◽

Thioredoxin Reductase ◽

Ros Generation ◽

Biosynthesis Pathway ◽

Cucumis Sativus L

In this study, the role of the antioxidant system has been examined in the regulation of the chlorophyll biosynthesis pathway in the vascular plant Cucumis sativus L. To generate reactive oxygen species (ROS), etiolated (E) and green (G) cucumber cotyledons were treated with methyl viologen (MV) or were exposed to high light (HL, 400–500 µE m–2 s–1). ROS generation was confirmed by measuring proline and H2O2 concentrations. With the effects of the MV- and HL-induced oxidative stress, it was observed that the chlorophyll biosynthesis pathway was severely affected in the HL-treated etiolated cotyledons (E-HL), MV-treated etiolated cotyledons (E-MV) and in MV-treated green cotyledons (G-MV) at 5-amino levulinic acid (ALA) as well as at protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester levels. The antioxidant assays conducted showed that the ascorbate peroxidase (APX) activity had decreased in the E-HL, E-MV and G-MV cotyledons along with the levels of ascorbate and lutein. A decrease in the NADPH-dependent thioredoxin reductase (NTRC) was also observed in the MV-treated cotyledons with a significant impairment of the catalase activity in the E-HL cotyledons. Conversely, in the HL-treated green i.e. G-HL cotyledons, where the accumulation of H2O2 and the inhibition of chlorophyll biosynthesis were not observed, an increase in the levels of APX, NTRC, peroxiredoxin, ascorbate, glutathione and lutein was noted. Thus, the results obtained suggested that the antioxidant system could influence the flow of the chlorophyll biosynthesis pathway through maintaining the levels of H2O2.

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Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

10.1101/2020.02.14.949297 ◽

2020 ◽

Author(s):

François Halloy ◽

Pavithra S. Iyer ◽

Paulina Ćwiek ◽

Alice Ghidini ◽

Jasmin Barman-Aksözen ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Bone Marrow Cells ◽

Therapeutic Benefit ◽

Biosynthesis Pathway ◽

Loss Of Function ◽

Erythropoietic Protoporphyria ◽

Aberrant Splicing ◽

Patients Experience ◽

Distinct Distribution

ABSTRACTErythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favours aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

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