In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

1st Faculty of Medicine Charles University Katerinska 32 Prague 12108 Czech Republic

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 25250 Czech Republic

Faculty of Science Charles University Vinicna 5 Prague 12108 Czech Republic

Laboratory of Bioenergetics Institute of Physiology of the Czech Academy of Sciences Videnska 1083 Prague 14220 Czech Republic

Laboratory of Cellular Metabolism Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 25250 Czech Republic

Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 25250 Czech Republic

Laboratory of Tumour Resistance Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 25250 Czech Republic

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Heinemann IU, Jahn M, Jahn D. The biochemistry of heme biosynthesis. Arch. Biochem. Biophys. 2008;474:238–251. doi: 10.1016/j.abb.2008.02.015. PubMed DOI

Phillips JD. Heme biosynthesis and the porphyrias. Mol. Genet. Metab. 2019;128:164–177. doi: 10.1016/j.ymgme.2019.04.008. PubMed DOI PMC

Hamza I, Dailey HA. One ring to rule them all: Trafficking of heme and heme synthesis intermediates in the metazoans. Biochim. Biophys. Acta. 1823;1617–1632:2012. doi: 10.1016/j.bbamcr.2012.04.009. PubMed DOI PMC

Swenson SA, et al. From synthesis to utilization: The ins and outs of mitochondrial heme. Cells. 2020 doi: 10.3390/cells9030579. PubMed DOI PMC

Chiabrando D, Vinchi F, Fiorito V, Mercurio S, Tolosano E. Heme in pathophysiology: A matter of scavenging, metabolism and trafficking across cell membranes. Front. Pharmacol. 2014 doi: 10.3389/fphar.2014.00061. PubMed DOI PMC

Ajioka RS, Phillips JD, Kushner JP. Biosynthesis of heme in mammals. Biochim. Biophys. Acta. 2006;1763:723–736. doi: 10.1016/j.bbamcr.2006.05.005. PubMed DOI

Consoli V, Sorrenti V, Grosso S, Vanella L. Heme oxygenase-1 signaling and redox homeostasis in physiopathological conditions. Biomolecules. 2021 doi: 10.3390/biom11040589. PubMed DOI PMC

Stocker R, Perrella MA. Heme oxygenase-1: A novel drug target for atherosclerotic diseases? Circulation. 2006;114:2178–2189. doi: 10.1161/CIRCULATIONAHA.105.598698. PubMed DOI

Hanna DA, Martinez-Guzman O, Reddi AR. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors. Biochemistry. 2017;56:1815–1823. doi: 10.1021/acs.biochem.7b00007. PubMed DOI PMC

Reddi AR, Hamza I. Heme mobilization in animals: A metallolipid's journey. Acc. Chem. Res. 2016;49:1104–1110. doi: 10.1021/acs.accounts.5b00553. PubMed DOI PMC

Duffy SP, et al. The Fowler syndrome-associated protein FLVCR2 is an importer of heme. Mol. Cell Biol. 2010;30:5318–5324. doi: 10.1128/MCB.00690-10. PubMed DOI PMC

Le Blanc S, Garrick MD, Arredondo M. Heme carrier protein 1 transports heme and is involved in heme-Fe metabolism. Am. J. Physiol. Cell Physiol. 2012;302:C1780–1785. doi: 10.1152/ajpcell.00080.2012. PubMed DOI

Rajagopal A, et al. Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins. Nature. 2008;453:1127–1131. doi: 10.1038/nature06934. PubMed DOI PMC

Ascenzi P, et al. Hemoglobin and heme scavenging. IUBMB Life. 2005;57:749–759. doi: 10.1080/15216540500380871. PubMed DOI

Kristiansen M, et al. Identification of the haemoglobin scavenger receptor. Nature. 2001;409:198–201. doi: 10.1038/35051594. PubMed DOI

Donegan RK, Moore CM, Hanna DA, Reddi AR. Handling heme: The mechanisms underlying the movement of heme within and between cells. Free Radic. Biol. Med. 2019;133:88–100. doi: 10.1016/j.freeradbiomed.2018.08.005. PubMed DOI PMC

Dailey HA, Meissner PN. Erythroid heme biosynthesis and its disorders. Cold Spring Harb. Perspect. Med. 2013;3:a011676. doi: 10.1101/cshperspect.a011676. PubMed DOI PMC

Arora S, Young S, Kodali S, Singal AK. Hepatic porphyria: A narrative review. Indian J. Gastroenterol. 2016;35:405–418. doi: 10.1007/s12664-016-0698-0. PubMed DOI

Stein PE, Badminton MN, Rees DC. Update review of the acute porphyrias. Br. J. Haematol. 2017;176:527–538. doi: 10.1111/bjh.14459. PubMed DOI

Yasuda M, Chen B, Desnick RJ. Recent advances on porphyria genetics: Inheritance, penetrance & molecular heterogeneity, including new modifying/causative genes. Mol. Genet. Metab. 2019;128:320–331. doi: 10.1016/j.ymgme.2018.11.012. PubMed DOI PMC

Brenner DA, Bloomer JR. The enzymatic defect in variegate porphyria. Studies with human cultured skin fibroblasts. N. Engl. J. Med. 1980;302:765–769. doi: 10.1056/NEJM198004033021401. PubMed DOI

Deybach JC, de Verneuil H, Nordmann Y. The inherited enzymatic defect in porphyria variegata. Hum. Genet. 1981;58:425–428. doi: 10.1007/BF00282829. PubMed DOI

Dailey HA, Dailey TA. Characteristics of human protoporphyrinogen oxidase in controls and variegate porphyrias. Cell. Mol. Biol. (Noisy-le-grand) 1997;43:67–73. PubMed

Frank J, Lam H, Zaider E, Poh-Fitzpatrick M, Christiano AM. Molecular basis of variegate porphyria: A missense mutation in the protoporphyrinogen oxidase gene. J. Med. Genet. 1998;35:244–247. doi: 10.1136/jmg.35.3.244. PubMed DOI PMC

Frank J, McGrath JA, Poh-Fitzpatrick MB, Hawk JL, Christiano AM. Mutations in the translation initiation codon of the protoporphyrinogen oxidase gene underlie variegate porphyria. Clin. Exp. Dermatol. 1999;24:296–301. doi: 10.1046/j.1365-2230.1999.00484.x. PubMed DOI

Wang B, et al. Quantitative structural insight into human variegate porphyria disease. J. Biol. Chem. 2013;288:11731–11740. doi: 10.1074/jbc.M113.459768. PubMed DOI PMC

Deybach JC, et al. Mutations in the protoporphyrinogen oxidase gene in patients with variegate porphyria. Hum. Mol. Genet. 1996;5:407–410. doi: 10.1093/hmg/5.3.407. PubMed DOI

Meissner PN, et al. A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria. Nat. Genet. 1996;13:95–97. doi: 10.1038/ng0596-95. PubMed DOI

Kirsch RE, Meissner PN, Hift RJ. Variegate porphyria. Semin. Liver Dis. 1998;18:33–41. doi: 10.1055/s-2007-1007138. PubMed DOI

Medlock AE, Meissner PN, Davidson BP, Corrigall AV, Dailey HA. A mouse model for South African (R59W) variegate porphyria: Construction and initial characterization. Cell Mol. Biol. (Noisy-le-grand) 2002;48:71–78. PubMed

Puy H, Robreau AM, Rosipal R, Nordmann Y, Deybach JC. Protoporphyrinogen oxidase: Complete genomic sequence and polymorphisms in the human gene. Biochem. Biophys. Res. Co. 1996;226:226–230. doi: 10.1006/bbrc.1996.1337. PubMed DOI

Roberts AG, et al. Partial characterization and assignment of the gene for protoporphyrinogen oxidase and variegate porphyria to human-chromosome 1q23. Hum. Mol. Genet. 1995;4:2387–2390. doi: 10.1093/hmg/4.12.2387. PubMed DOI

Taketani S, et al. The human protoporphyrinogen oxidase gene (PPOX): Organization and location to chromosome 1. Genomics. 1995;29:698–703. doi: 10.1006/geno.1995.9949. PubMed DOI

Beale, S. I. & Weinstein, J. D. Biosynthesis of Heme and Chlorophylls. (ed. Dailey, H. A.). 287–391 (McGraw-Hill, 1990).

Dailey TA, Dailey HA. Human protoporphyrinogen oxidase: Expression, purification, and characterization of the cloned enzyme. Protein Sci. 1996;5:98–105. doi: 10.1002/pro.5560050112. PubMed DOI PMC

Akhtar M. The modification of acetate and propionate side-chains during the biosynthesis of heme and chlorophylls—Mechanistic and stereochemical studies. Ciba F Symp. 1994;180:131–151. PubMed

Nishimura K, Taketani S, Inokuchi H. Cloning of a human cDNA for protoporphyrinogen oxidase by complementation in vivo of a hemG mutant of Escherichia coli. J. Biol. Chem. 1995;270:8076–8080. doi: 10.1074/jbc.270.14.8076. PubMed DOI

Medlock AE, et al. Identification of the mitochondrial heme metabolism complex. PLoS ONE. 2015;10:e0135896. doi: 10.1371/journal.pone.0135896. PubMed DOI PMC

Rhee HW, et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science. 2013;339:1328–1331. doi: 10.1126/science.1230593. PubMed DOI PMC

Dooley KA, et al. Montalcino, A zebrafish model for variegate porphyria. Exp. Hematol. 2008;36:1132–1142. doi: 10.1016/j.exphem.2008.04.008. PubMed DOI PMC

Dayan FE, Duke SO. Protoporphyrinogen oxidase-inhibiting herbicides. Hayes' Handb. Pestic. Toxicol. 2010;3:1733–1751. doi: 10.1016/B978-0-12-374367-1.00081-1. DOI

Theodoridis G, Liebl R, Zagar C. Protoporphyrinogen IX oxidase inhibitors. Mod. Crop Protect. Compds. 2012;2:163–195. doi: 10.1002/9783527644179.ch3. DOI

Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46:W242–W245. doi: 10.1093/nar/gky354. PubMed DOI PMC

Al-Romaih K, et al. Chromosomal instability in osteosarcoma and its association with centrosome abnormalities. Cancer Genet. Cytogenet. 2003;144:91–99. doi: 10.1016/s0165-4608(02)00929-9. PubMed DOI

Raftopoulou C, et al. Karyotypic flexibility of the complex cancer genome and the role of polyploidization in maintenance of structural integrity of cancer chromosomes. Cancers (Basel) 2020 doi: 10.3390/cancers12030591. PubMed DOI PMC

Marcero JR, PielIii RB, Burch JS, Dailey HA. Rapid and sensitive quantitation of heme in hemoglobinized cells. Biotechniques. 2016;61:83–91. doi: 10.2144/000114444. PubMed DOI

Sohoni S, et al. Elevated heme synthesis and uptake underpin intensified oxidative metabolism and tumorigenic functions in non-small cell lung cancer cells. Cancer Res. 2019;79:2511–2525. doi: 10.1158/0008-5472.CAN-18-2156. PubMed DOI

Pereira TF, et al. Fluorescence-based method is more accurate than counting-based methods for plotting growth curves of adherent cells. BMC Res. Notes. 2020;13:57. doi: 10.1186/s13104-020-4914-8. PubMed DOI PMC

Cable EE, Miller TG, Isom HC. Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: Delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms. Arch. Biochem. Biophys. 2000;384:280–295. doi: 10.1006/abbi.2000.2117. PubMed DOI

Lathrop JT, Timko MP. Regulation by heme of mitochondrial protein transport through a conserved amino acid motif. Science. 1993;259:522–525. doi: 10.1126/science.8424176. PubMed DOI

Munakata H, et al. Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase. J. Biochem. 2004;136:233–238. doi: 10.1093/jb/mvh112. PubMed DOI

Hamilton JW, et al. Heme regulates hepatic 5-aminolevulinate synthase mRNA expression by decreasing mRNA half-life and not by altering its rate of transcription. Arch. Biochem. Biophys. 1991;289:387–392. doi: 10.1016/0003-9861(91)90428-l. PubMed DOI

Sassa S, Granick S. Induction of -aminolevulinic acid synthetase in chick embryo liver cells in cluture. Proc. Natl. Acad. Sci. U S A. 1970;67:517–522. doi: 10.1073/pnas.67.2.517. PubMed DOI PMC

Tian Q, et al. Lon peptidase 1 (LONP1)-dependent breakdown of mitochondrial 5-aminolevulinic acid synthase protein by heme in human liver cells. J. Biol. Chem. 2011;286:26424–26430. doi: 10.1074/jbc.M110.215772. PubMed DOI PMC

Yamamoto M, Hayashi N, Kikuchi G. Evidence for the transcriptional inhibition by heme of the synthesis of delta-aminolevulinate synthase in rat liver. Biochem. Biophys. Res. Commun. 1982;105:985–990. doi: 10.1016/0006-291x(82)91067-1. PubMed DOI

Yamamoto M, Hayashi N, Kikuchi G. Translational inhibition by heme of the synthesis of hepatic delta-aminolevulinate synthase in a cell-free system. Biochem. Biophys. Res. Commun. 1983;115:225–231. doi: 10.1016/0006-291x(83)90993-2. PubMed DOI

Dailey TA, Woodruff JH, Dailey HA. Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase. Biochem. J. 2005;386:381–386. doi: 10.1042/BJ20040570. PubMed DOI PMC

Jerico D, et al. mRNA-based therapy in a rabbit model of variegate porphyria offers new insights into the pathogenesis of acute attacks. Mol. Ther. Nucleic Acids. 2021;25:207–219. doi: 10.1016/j.omtn.2021.05.010. PubMed DOI PMC

Hao GF, Zuo Y, Yang SG, Yang GF. Protoporphyrinogen oxidase inhibitor: An ideal target for herbicide discovery. Chimia (Aarau) 2011;65:961–969. doi: 10.2533/chimia.2011.961. PubMed DOI

Jacobs JM, Jacobs NJ, Sherman TD, Duke SO. Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley. Plant Physiol. 1991;97:197–203. doi: 10.1104/pp.97.1.197. PubMed DOI PMC

Arnould S, Camadro JM. The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. Proc. Natl. Acad. Sci. USA. 1998;95:10553–10558. doi: 10.1073/pnas.95.18.10553. PubMed DOI PMC

Witkowski DA, Halling BP. Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl. Plant Physiol. 1989;90:1239–1242. doi: 10.1104/pp.90.4.1239. PubMed DOI PMC

Quest JA, et al. Evaluation of the carcinogenic potential of pesticides. 1. Acifluorfen. Regul. Toxicol. Pharmacol. 1989;10:149–159. doi: 10.1016/0273-2300(89)90022-6. PubMed DOI

Matringe M, Camadro JM, Labbe P, Scalla R. Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides. Biochem. J. 1989;260:231–235. doi: 10.1042/bj2600231. PubMed DOI PMC

Jakubek M, et al. PPO-inhibiting herbicides and structurally relevant Schiff bases: Evaluation of inhibitory activities against human protoporphyrinogen oxidase. Processes. 2021 doi: 10.3390/pr9020383. DOI

Frank J, et al. Homozygous variegate porphyria: Identification of mutations on both alleles of the protoporphyrinogen oxidase gene in a severely affected proband. J. Invest. Dermatol. 1998;110:452–455. doi: 10.1046/j.1523-1747.1998.00148.x. PubMed DOI

Dixon N, et al. Pilot study of mitochondrial bioenergetics in subjects with acute porphyrias. Mol. Genet. Metab. 2019;128:228–235. doi: 10.1016/j.ymgme.2019.05.010. PubMed DOI PMC

Ran FA, et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013;8:2281–2308. doi: 10.1038/nprot.2013.143. PubMed DOI PMC

Shepherd M, Dailey HA. A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation. Anal. Biochem. 2005;344:115–121. doi: 10.1016/j.ab.2005.06.012. PubMed DOI PMC

Fyrestam J, Ostman C. Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli. Anal. Bioanal. Chem. 2017;409:6999–7010. doi: 10.1007/s00216-017-0610-5. PubMed DOI PMC

Beika M, et al. Accumulation of uroporphyrin I in necrotic tissues of squamous cell carcinoma after administration of 5-aminolevulinic acid. Int. J. Mol. Sci. 2021 doi: 10.3390/ijms221810121. PubMed DOI PMC

Hartmannova H, et al. Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6. Hum. Mol. Genet. 2016;25:4062–4079. doi: 10.1093/hmg/ddw245. PubMed DOI

Tyanova S, et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Wittig I, Braun HP, Schagger H. Blue native PAGE. Nat. Protoc. 2006;1:418–428. doi: 10.1038/nprot.2006.62. PubMed DOI

Cunatova K, et al. Loss of COX4I1 leads to combined respiratory chain deficiency and impaired mitochondrial protein synthesis. Cells. 2021 doi: 10.3390/cells10020369. PubMed DOI PMC

PajueloReguera D, et al. Cytochrome c oxidase subunit 4 isoform exchange results in modulation of oxygen affinity. Cells. 2020 doi: 10.3390/cells9020443. PubMed DOI PMC

Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat. Methods. 2012;9:671–675. doi: 10.1038/nmeth.2089. PubMed DOI PMC