ABSTRACT
Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.
Subject(s)
Drug Design , Enzyme Inhibitors , Herbicides , Nicotiana , Plant Proteins , Protoporphyrinogen Oxidase , Pyridazines , Protoporphyrinogen Oxidase/antagonists & inhibitors , Protoporphyrinogen Oxidase/metabolism , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/genetics , Pyridazines/chemistry , Pyridazines/pharmacology , Herbicides/pharmacology , Herbicides/chemistry , Herbicides/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Structure-Activity Relationship , Nicotiana/metabolism , Nicotiana/enzymology , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Molecular Docking Simulation , Molecular Structure , Plant Weeds/drug effects , Plant Weeds/enzymology , KineticsABSTRACT
Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.
Subject(s)
Herbicides , Plant Proteins , Protoporphyrinogen Oxidase , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolism , Herbicides/pharmacology , Plant Proteins/metabolism , Plant Proteins/genetics , Plastids/genetics , Plastids/metabolism , Gene Expression Regulation, Plant , Amaranthus/genetics , Amaranthus/drug effects , Chloroplasts/metabolism , Chloroplasts/genetics , Herbicide Resistance/genetics , Arabidopsis/genetics , Thylakoids/metabolismABSTRACT
Palmer amaranth has evolved target and nontarget site resistance to protoporphyrinogen oxidase-inhibitor herbicides in the United States. Recently, a population (KCTR) from a long-term conservation tillage study in Kansas was found to be resistant to herbicides from six sites of action, including to PPO-inhibitors, even with this herbicide group being minimally used in this field. This research investigated the level of resistance to postemergence PPO-inhibitors, target- and nontarget-site resistance mechanism(s), and efficacy of pre-emergence chemistries. The greenhouse experiments confirmed 6.1- to 78.9-fold resistance to lactofen in KCTR, with the level of resistance increasing when KCTR was purified for the resistance trait. PPO2 sequences alignment revealed the absence of known mutations conferring resistance to PPO-inhibitors in KCTR Palmer amaranth, and differential expression of the PPO2 gene did not occur. KCTR metabolized fomesafen faster than the susceptible population, indicating that herbicide detoxification is the mechanism conferring resistance in this population. Further, treatment with the cytochrome P450-inhibitor malathion followed by lactofen restored the sensitivity of KCTR to this herbicide. Despite being resistant to POST applied PPO-inhibitors, KCTR Palmer amaranth was completely controlled by the labeled rate of the PRE applied PPO-inhibitors fomesafen, flumioxazin, saflufenacil, sulfentrazone, and oxadiazon. The overall results suggest that P450-mediated metabolism confers resistance to PPO-inhibitors in KCTR, rather than alterations in the PPO2, which were more commonly found in other Palmer amaranth populations. Future work will focus on identifying the fomesafen metabolites and on unravelling the genetic basis of metabolic resistance to PPO-inhibitor herbicides in KCTR Palmer amaranth.
Subject(s)
Amaranthus , Benzamides , Halogenated Diphenyl Ethers , Herbicides , Herbicides/pharmacology , Kansas , Protoporphyrinogen Oxidase/genetics , Herbicide Resistance/genetics , Amaranthus/metabolismABSTRACT
BACKGROUND: Variegate porphyria is caused by mutations in the PPOX gene; it usually presents in adolescents and adults as an autosomal dominant condition, with cutaneous features or acute peripheral and/or central nervous system crises. A rarer variant, homozygous variegate porphyria, presents in childhood with cutaneous manifestations as well as neurophenotypes. This study sought to further characterize the homozygous PPOX-related neuroendocrine phenotype. METHODS: This study is a retrospective review of the patients' charts, including their clinical evaluation and molecular genetics, neurodiagnostic, and neuroradiological investigations. RESULTS: We describe here three children from a consanguineous family who presented with nystagmus, developmental delay and ataxia, photosensitive skin manifestations, and adrenal insufficiency. Analysis of porphyrins in plasma, urine, and stool together with a genetic study of the PPOX gene confirmed the diagnosis. Interestingly, brain MRI showed severe hypomyelination, a finding rarely reported in variegate porphyria, together with adrenal insufficiency. CONCLUSION: We recommend analysis of porphyrins in unexplained hypomyelination disorders. Patients with variegate porphyria should be tested for adrenal insufficiency.
Subject(s)
Adrenal Insufficiency , Porphyria, Variegate , Porphyrins , Child , Humans , Adrenal Insufficiency/complications , Flavoproteins/genetics , Mitochondrial Proteins/genetics , Phenotype , Porphyria, Variegate/genetics , Porphyria, Variegate/complications , Protoporphyrinogen Oxidase/geneticsABSTRACT
BACKGROUND AND AIM: Gene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis. PATIENTS AND METHODS: Both paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families. RESULTS: WES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations. CONCLUSION: Our findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.
Subject(s)
Cholestasis, Intrahepatic , Cholestasis , Humans , Mutation , Exome Sequencing , Cholestasis/genetics , Genotype , Cholestasis, Intrahepatic/genetics , Cholestasis, Intrahepatic/diagnosis , Flavoproteins/genetics , Mitochondrial Proteins/genetics , Protoporphyrinogen Oxidase/genetics , Cytoskeletal Proteins/genetics , Adaptor Proteins, Signal Transducing/geneticsABSTRACT
There are several methods to control weeds, which impose particular challenges for farmers in all parts of the world, although applying small molecular compounds still remains the most efficient technology to date. However, plants can evolve to become resistant toward active ingredients which is also the case for protoporphyrinogen oxidase (PPO) inhibitors, a class of highly effective herbicides in use for more than 50 years. Hence, it is essential to continuously discover and develop new herbicidal PPO inhibitors with enhanced intrinsic activity, an improved resistance profile, enhanced crop safety, favorable physicochemical properties, and a clean toxicological profile. By modifying structural key features from known PPO inhibitors such as tiafenacil, inspired by isostere and mix&match concepts in combination with modeling investigations based on a wild-type Amaranthus crystal structure, we have found new promising lead structures showing strong activity in vitro and in vivo against several notorious dicotyledon and monocotyledon weeds with emerging resistance (e.g., Amaranthus palmeri, Amaranthus tuberculatus, Lolium rigidum, and Alopecurus myosuroides). While several phenyl uracils carrying an isoxazoline motif in their thio-linked side chain showed promising resistance-breaking potential against different Amaranthus species, introducing a thioacrylamide side chain afforded outstanding efficacy against resistant grass weeds.
Subject(s)
Amaranthus , Herbicides , Magnoliopsida , Protoporphyrinogen Oxidase/genetics , Herbicides/pharmacology , Plant Weeds , Poaceae , Herbicide ResistanceABSTRACT
Myelodysplastic syndromes (MDS) are acquired bone marrow malignant disorders characterized by ineffective hematopoiesis, resulting from a complex interaction between genetic and epigenetic mutations, alterations of the marrow microenvironment, and the immune system. In 2001, the World Health Organization (WHO) proposed a classification that integrates morphologic and genetic information, considering the MDS with ring sideroblasts (MDS-RS) as a distinct entity. Considering the strong association between MDS-RS and SF3B1 mutation and its importance in the development of MDS, the last WHO classification replaced the prior entity of MDS-RS with MDS with SF3B1 mutation. Several studies were performed to explore this genotype-phenotype correlation. Mutant SF3B1 protein deregulates the expression of genes implicated in developing hematopoietic stem and progenitor cells. Of paramount importance are PPOX and ABCB7 involved in iron metabolism. Another essential role in hemopoiesis is played by the transforming growth factor-beta (TGF-ß) receptor. This gene exerts its effects on SMAD pathways, regulating hematopoiesis through effects on balancing proliferation and apoptosis cell inactivity, differentiation, and migration. Luspatercept (ACE-536) is a soluble fusion protein that inhibits molecules in the TGF-ß superfamily. Since its structure resembles the TGF-ß family receptor, it catches TGF-ß superfamily ligands before binding to the receptor, resulting in reduced activation of SMAD signaling, thus enabling erythroid maturation. Luspatercept was investigated in the phase III trial MEDALIST, showing promising efficacy in treating anemia compared to placebo. Nowadays, further studies are needed to explore the real potential of luspatercept, investigating the biological features likely associated with treatment response, the potential use in combination treatments, and its role in the treatment of naïve MDS.
Subject(s)
Anemia , Myelodysplastic Syndromes , Humans , RNA Splicing Factors/genetics , Myelodysplastic Syndromes/drug therapy , Bone Marrow/pathology , Mutation , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/therapeutic use , Phosphoproteins/genetics , Phosphoproteins/therapeutic use , Flavoproteins/genetics , Flavoproteins/therapeutic use , Mitochondrial Proteins/genetics , Mitochondrial Proteins/therapeutic use , Protoporphyrinogen Oxidase/geneticsABSTRACT
All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified 2 ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the 2 isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. Mitochondrial localization of PPO2 in Arabidopsis was ruled out. Since Arabidopsis PPO2 does not encode a cleavable transit peptide, integration of the protein into the chloroplast envelope must make use of a noncanonical import route. However, when a chloroplast transit peptide was fused to the N-terminus of PPO2, the enzyme was detected predominantly in thylakoid membranes and was able to fully complement ppo1. Thus, the 2 PPO isoforms in Arabidopsis are functionally equivalent but spatially separated. Their distinctive localizations within plastids thus enable the synthesis of discrete subpools of the PPO product protoporphyrin IX, which may serve different cellular needs.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chloroplasts/metabolism , Plastids/metabolism , Protein Isoforms/genetics , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolismABSTRACT
The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a ΔhemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.
Subject(s)
Cyanobacteria , Escherichia coli , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolism , Escherichia coli/genetics , Phylogeny , Cyanobacteria/genetics , Heme/metabolism , Chlorophyll/metabolismABSTRACT
INTRODUCTION: Acute intermittent porphyria (AIP) is a very rare (orphan) metabolic disorder of porphyrin biosynthesis which is characterized by elevated plasma and urine levels of 5-aminolevulinic acid (5-ALA) and porphobilinogen (PBG). Patients with this disorder which is caused by a germline mutation of the hydroxymethylbilan-synthase (HMBS)-gene have a high risk of primary liver cancer which may be determined by disease activity. The exact mechanism of carcinogenesis of this rare tumor is unknown, however. MATERIALS AND METHODS: We analyzed paraffin-embedded formalin-fixed liver tumor and normal liver specimens of two female AIP patients treated at the Munich EPNET center. One patient had developed hepatocellular carcinoma (HCC), the other intrahepatic cholangiocarcinoma (CCA). Since biallelic inactivation of HMBS had been observed in one study, we used Sanger and next-generation sequencing with a 8 gene porphyria panel plus 6 potential modifier loci to search for mutations in DNA extractions. RESULTS: In the patient with the HCC, we found a second inactivating mutation in the HMBS gene in the tumor but not in the adjacent normal liver tissue. No mutation could be found in the liver tissues of the patient with CCA, however. CONCLUSIONS: Biallelic inactivation of HMBS or protoporphyrinogen-oxidase (PPOX), another enzyme of porphyrin biosynthesis, has been observed in patients with acute porphyrias and liver tumors. We could confirm this in our patient with HCC with a mutation in HMBS but not in the one with CCA. Since 5-ALA can be converted into carcinogenic substances such as 4,5-dioxovaleric acid (DOVA) or 3,6-dihydropyrazine-2,5-dipropanoic acid (= cyclic dimerization product of 5-ALA), local production of these metabolites in hepatic areas with complete loss of HMBS activity may contribute to liver carcinogenesis.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Porphyria, Acute Intermittent , Porphyrins , Female , Humans , Aminolevulinic Acid/urine , Carcinogenesis , Carcinoma, Hepatocellular/genetics , Flavoproteins , Liver Neoplasms/genetics , Mitochondrial Proteins , Porphyria, Acute Intermittent/genetics , Porphyria, Acute Intermittent/pathology , Protoporphyrinogen Oxidase/genetics , AdultABSTRACT
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.
Subject(s)
Oxidoreductases , Porphyria, Variegate , Aminolevulinic Acid/metabolism , CRISPR-Cas Systems , Cell Line , Heme , Humans , Oxidoreductases/genetics , Oxidoreductases/metabolism , Porphyria, Variegate/genetics , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolism , ProtoporphyrinsABSTRACT
Resistance to protoporphyrinogen IX oxidase (PPO)-inhibitors in Amaranthus palmeri and Amaranthus tuberculatus is mainly contributed by mutations in the PPO enzyme, which renders herbicide molecules ineffective. The deletion of glycine210 (ΔG210) is the most predominant PPO mutation. ΔG210-ppo2 is overexpressed in rice (Oryza sativa c. 'Nipponbare') and Arabidopsis thaliana (Col-0). A foliar assay was conducted on transgenic T1 rice plants with 2× dose of fomesafen (780 g ha−1), showing less injury than the non-transgenic (WT) plants. A soil-based assay conducted with T2 rice seeds confirmed tolerance to fomesafen applied pre-emergence. In agar medium, root growth of WT rice seedlings was inhibited >90% at 5 µM fomesafen, while root growth of T2 seedlings was inhibited by 50% at 45 µM fomesafen. The presence and expression of the transgene were confirmed in the T2 rice survivors of soil-applied fomesafen. A soil-based assay was also conducted with transgenic A. thaliana expressing ΔG210-ppo2 which confirmed tolerance to the pre-emergence application of fomesafen and saflufenacil. The expression of A. palmeri ΔG210-ppo2 successfully conferred tolerance to soil-applied fomesafen in rice and Arabidopsis. This mutant also confers cross-tolerance to saflufenacil in Arabidopsis. This trait could be introduced into high-value crops that lack chemical options for weed management.
Subject(s)
Amaranthus , Arabidopsis , Oryza , Amaranthus/genetics , Arabidopsis/genetics , Arabidopsis/metabolism , Herbicide Resistance/genetics , Oryza/genetics , Oryza/metabolism , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolism , SoilABSTRACT
Protoporphyrinogen IX (Protogen IX) oxidase (PPO) catalyzes the oxidation of Protogen IX to Proto IX. PPO is also the target site for diphenyl ether-type herbicides. In plants, there are two PPO encoding genes, PPO1 and PPO2. To date, no PPO gene or mutant has been characterized in monocotyledonous plants. In this study, we isolated a spotted and rolled leaf (sprl1) mutant in rice (Oryza sativa). The spotted leaf phenotype was sensitive to high light intensity and low temperature, but the rolled leaf phenotype was insensitive. We confirmed that the sprl1 phenotypes were caused by a single nucleotide substitution in the OsPPO1 (LOC_Os01g18320) gene. This gene is constitutively expressed, and its encoded product is localized to the chloroplast. The sprl1 mutant accumulated excess Proto(gen) IX and reactive oxygen species (ROS), resulting in necrotic lesions. The expressions of 26 genes associated with tetrapyrrole biosynthesis, photosynthesis, ROS accumulation, and rolled leaf were significantly altered in sprl1, demonstrating that these expression changes were coincident with the mutant phenotypes. Importantly, OsPPO1-overexpression transgenic plants were resistant to the herbicides oxyfluorfen and acifluorfen under field conditions, while having no distinct influence on plant growth and grain yield. These finding indicate that the OsPPO1 gene has the potential to engineer herbicide resistance in rice.
Subject(s)
Herbicides , Oryza , Herbicide Resistance/genetics , Herbicides/pharmacology , Mutation , Oryza/genetics , Oryza/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolism , Reactive Oxygen SpeciesABSTRACT
BACKGROUND: Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides is endowed primarily by target-site mutations at the PPX2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus spp. PPX2, the most prevalent target mutations are deletion of the G210 codon, and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket, resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same PPX2 allele. RESULTS: Plants carrying R128G+/+ ΔG210+/-, where + indicates presence of the mutation, were crossed with each other. The PPX2 of the offspring was subjected to pyrosequencing and E. coli-based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur in one allele only; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. CONCLUSIONS: Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Subject(s)
Amaranthus , Herbicides , Alleles , Amaranthus/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Herbicide Resistance/genetics , Herbicides/pharmacology , Mutation , Protoporphyrinogen Oxidase/geneticsABSTRACT
Variegate Porphyria (VP) is an inherited rare disorder that is caused by mutations in the protoporphyrinogen oxidase (PPOX) gene. This deficiency is associated with the accumulation of porphyrins and porphyrin precursors in the body, which, in turn, can potentially result in a variety of skin and neurological symptoms. Here, we reported a 7-year-old boy with homozygous VP and novel mutation on PPOX gene. He was admitted with three episodes of generalized tonic-clonic seizure in the last 6 months. He was presented with lesions, hyperpigmentation, fragility, and blistering of sun-exposed skin. The weakness of limbs and brachydactyly were observed. In the follow-up, he had aggressive behavior, learning disability and abdominal pain, particularly around the navel. Eventually, the whole exome sequencing (WES) result reported a novel homozygous pathogenic variant (c.1072G > A p.G358R) in PPOX gene which confirmed the VP. He had been advised to be away from the sun and use sunscreen regularly.
Subject(s)
Porphyria, Variegate , Child , Fingers , Flavoproteins/genetics , Humans , Iran , Male , Mitochondrial Proteins/genetics , Mutation , Porphyria, Variegate/diagnosis , Porphyria, Variegate/genetics , Protoporphyrinogen Oxidase/geneticsABSTRACT
SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of â¼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.
Subject(s)
Mitochondrial Membrane Transport Proteins/metabolism , Myelodysplastic Syndromes , Phosphoproteins , ATP-Binding Cassette Transporters , Cell Differentiation/genetics , Flavoproteins/genetics , Flavoproteins/metabolism , Humans , Mitochondrial Proteins/genetics , Mutation , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Phosphoproteins/genetics , Protoporphyrinogen Oxidase/genetics , Protoporphyrinogen Oxidase/metabolism , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolismABSTRACT
Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.
Subject(s)
Flavoproteins/genetics , Mitochondrial Proteins/genetics , Mutation/genetics , Protoporphyrinogen Oxidase/genetics , Amino Acid Sequence , Biophysical Phenomena , Cell Line , Enzyme Stability , Flavoproteins/chemistry , Flavoproteins/isolation & purification , Humans , Kinetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/isolation & purification , Models, Molecular , Protein Multimerization , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/isolation & purification , Subcellular Fractions/metabolism , TemperatureABSTRACT
Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.
Subject(s)
Flavoproteins/biosynthesis , Flavoproteins/isolation & purification , Mitochondrial Proteins/biosynthesis , Mitochondrial Proteins/isolation & purification , Protoporphyrinogen Oxidase/biosynthesis , Protoporphyrinogen Oxidase/isolation & purification , Animals , Cell Line , Escherichia coli/genetics , Flavoproteins/genetics , HEK293 Cells , Humans , Mitochondrial Proteins/genetics , Protoporphyrinogen Oxidase/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Sf9 CellsABSTRACT
Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In human, point mutations on PPO are responsible for the dominantly inherited disorder disease, Variegate Porphyria (VP). Of the VP-causing mutation site, the Arg59 is by far the most prevalent VP mutation residue identified. Multiple sequences alignment of PPOs shows that the Arg59 of human PPO (hPPO) is not conserved, and experiments have shown that the equivalent residues in PPO from various species are essential for enzymatic activity. In this work, it was proposed that the Arg59 performs its function by forming a hydrogen-bonding (HB) network around it in hPPO, and we investigated the role of the HB network via site-directed mutagenesis, enzymatic kinetics and computational studies. We found the integrity of the HB network around Arg59 is important for enzyme activity. The HB network maintains the substrate binding chamber by holding the side chain of Arg59, while it stabilizes the micro-environment of the isoalloxazine ring of FAD, which is favorable for the substrate-FAD interaction. Our result provides a new insight to understanding the relationship between the structure and function for hPPO that non-conserved residues can form a conserved element to maintain the function of protein.
Subject(s)
Arginine/chemistry , Arginine/metabolism , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Amino Acid Sequence , Arginine/genetics , Enzyme Assays/methods , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Mutagenesis, Site-Directed/methods , Protein Structural Elements , Protoporphyrinogen Oxidase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Structure-Activity RelationshipABSTRACT
BACKGROUND: Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate- and acetolactate synthase-resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid-south and determined its correlation with the known functional PPO2 target-site mutations (TSM). RESULTS: The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high-survival-high-injury category, manifesting an early-stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. CONCLUSIONS: Resistance to PPO inhibitors is widespread across the Mid-southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co-occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co-occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co-occur in the same ppo2 allele.